U.S. patent number 10,457,953 [Application Number 15/486,841] was granted by the patent office on 2019-10-29 for tobacco plants exhibiting altered photosynthesis and methods of making and using.
This patent grant is currently assigned to Altria Client Services LLC. The grantee listed for this patent is Altria Client Services LLC. Invention is credited to Yanxin Shen, Dongmei Xu.
![](/patent/grant/10457953/US10457953-20191029-C00001.png)
![](/patent/grant/10457953/US10457953-20191029-C00002.png)
![](/patent/grant/10457953/US10457953-20191029-C00003.png)
![](/patent/grant/10457953/US10457953-20191029-C00004.png)
![](/patent/grant/10457953/US10457953-20191029-C00005.png)
![](/patent/grant/10457953/US10457953-20191029-C00006.png)
![](/patent/grant/10457953/US10457953-20191029-D00001.png)
![](/patent/grant/10457953/US10457953-20191029-D00002.png)
![](/patent/grant/10457953/US10457953-20191029-D00003.png)
![](/patent/grant/10457953/US10457953-20191029-D00004.png)
![](/patent/grant/10457953/US10457953-20191029-D00005.png)
View All Diagrams
United States Patent |
10,457,953 |
Shen , et al. |
October 29, 2019 |
Tobacco plants exhibiting altered photosynthesis and methods of
making and using
Abstract
This disclosure provides tobacco plants that exhibit altered
photosynthesis as well as methods of making and using such
plants.
Inventors: |
Shen; Yanxin (Glen Allen,
VA), Xu; Dongmei (Glen Allen, VA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Altria Client Services LLC |
Richmond |
VA |
US |
|
|
Assignee: |
Altria Client Services LLC
(Richmond, VA)
|
Family
ID: |
58632665 |
Appl.
No.: |
15/486,841 |
Filed: |
April 13, 2017 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20170298376 A1 |
Oct 19, 2017 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62322001 |
Apr 13, 2016 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/825 (20130101); C07K 14/415 (20130101); C12N
9/0073 (20130101); C12N 15/8261 (20130101); A24B
13/00 (20130101); A01H 1/06 (20130101); C12N
15/8251 (20130101); A01H 5/12 (20130101); C12N
15/8269 (20130101); C12Y 114/13122 (20130101); C12N
15/8218 (20130101); Y02A 40/146 (20180101) |
Current International
Class: |
C12N
15/82 (20060101); A24B 13/00 (20060101); A01H
5/12 (20180101); A01H 1/06 (20060101); C07K
14/415 (20060101); C12N 9/02 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Kirst, H. et al.; Planta, 2017, vol. 245, pp. 1009-1020. cited by
examiner .
Fred Lunt, 5 Things You Need to Know About PA Broadleaf Tobacco
(Aug. 15, 2018)
https---www.famous-smoke.com/cigaradvisor/5-things-about-pennsylvan-
ia-broadleaf-tobacco pp. 1-12. cited by examiner .
Yang, Y. et al. Journal of Experimental Botany, 2016, vol. 67, No.
5 pp. 1297-1310. cited by examiner .
Okabe, K. et al. Plant Physiology; 1977, vol. 60, pp. 150-156.
cited by examiner .
Arnon, Plant Physiol 24:1-5, 1949. cited by applicant .
Askura et al., "Non-identical contributions of two membrane-bound
cpSRP components, cpFtsY and Alb3, to thylakoid biogenesis," The
Plant Journal 56(6):1007-1017, Dec. 1, 2008. cited by applicant
.
Chenna et al., Nucleic Acids Res 31(13):3497-3500, 2003. cited by
applicant .
Dayhoff et al., Atlas of Protein Sequence and Structure (Suppl.
3):345-352, 1978. cited by applicant .
Henning et al., "The chloroplast signal recognition particle
(CpSRP) pathway as a tool to minimize chlorophyll antenna size and
maximize photosynthetic productivity," Biotechnology Advances
32(1):66-72, Feb. 28, 2014. cited by applicant .
Horsch et al., Science 227:1229-12231, 1985. cited by applicant
.
Kirst et al., "Assembly of the Light-Harvesting Chlorophyll Antenna
in the Green Alga Chlamdomans reinhartii Requires Expression of the
TLA2-CpFTSY Gene," Plant Physiology 158(2:930-945, Feb. 1, 2012.
cited by applicant .
Kirst et al., "Truncated Photosystem Chlorophyll Antenna Size in
the Green Microalga Chlamydomonas reinhardtii upon Deletion of the
TLA3-CpSRP43 Gene," Plant Physiol 160:2251-2260, 2012. cited by
applicant .
Kugelmann et al., "Phenotypes of Alb3p and carotenoid synthesis
mutants show similarities regarding light sensitivity, thylakoid
structure and protein stability," Photosynthetica 51(1):45-54, Mar.
1, 2013. cited by applicant .
Li et al., Nucleic Acids Res 39(14):6315-6325, 2011. cited by
applicant .
Mayo et al., Nat Protoc 1(3):1105-1111, 2006. cited by applicant
.
Melis et al., "New vistas in measurement of
photosynthesis--Spectroscopic methods in photosyntheses:
photosystem stoichiometry and chlorophyll antenna size," Philos
Trans R Soc Lond B 323:397-409, 1989. cited by applicant .
Polle et al., "tlal, a DNA insertional transformant of the green
alga Chlamydomonas reinhardtii with a truncated light-harvesting
chlorophyll antenna size," Planta 217:49-59, 2003. cited by
applicant .
Wright et al., "High-frequency homologous recombination in plants
mediated by zinc-finger nucleases," The Plant J 44:693-705, 2005.
cited by applicant.
|
Primary Examiner: Kallis; Russell
Attorney, Agent or Firm: Marsh; David R. Arnold & Porter
Kaye Scholer LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority under 35 U.S.C.
.sctn. 119(e) to U.S. Application No. 62/322,001 filed Apr. 13,
2016.
Claims
What is claimed is:
1. A method of making a Nicotiana tabacum plant, comprising:
inducing mutagenesis in N. tabacum cells to produce mutagenized N.
tabacum cells; obtaining one or more N. tabacum plants from said
mutagenized N. tabacum cells; and identifying at least one of said
N. tabacum plants that comprises a mutation in a sequence having at
least 95% sequence identity to SEQ ID NO:15 or 17.
2. The method of claim 1, further comprising identifying at least
one of said N. tabacum plants that comprises a reduced amount of
mRNA corresponding to SEQ ID NO:15 or 17; a reduced amount of a
polypeptide comprising SEQ ID NO:16 or 18; reduced activity of a
polypeptide comprising SEQ ID NO:16 or 18; a reduced concentration
of thylakoid membranes in the photosystems; a reduced amount of
total chlorophyll; an increased ratio of chlorophyll a to
chlorophyll b; an increased biomass relative to a N. tabacum plant
lacking a mutated polynucleotide having a sequence of SEQ ID NO:15
or 17; or a combination thereof.
3. The method of claim 1, wherein leaf from the mutant N. tabacum
plant exhibits comparable or better quality than leaf from the
plant lacking said mutation.
4. The method of claim 1, wherein said N. tabacum plant is a Burley
type, a dark type, a flue-cured type, or an Oriental type.
5. A Nicotiana tabacum plant, or part thereof, comprising a
mutation in an endogenous nucleic acid, the wild type endogenous
nucleic acid encoding a sequence having at least 95% sequence
identity to a sequence selected from the group consisting of SEQ ID
NOs: 16 and 18.
6. The N. tabacum plant, or part thereof, of claim 5, wherein leaf
from a mutant plant comprises a reduced amount of mRNA
corresponding to a sequence selected from the group consisting of
SEQ ID NOs:15 and 17; a reduced amount of a polypeptide selected
from the group consisting of SEQ ID NOs:16 and 18; reduced activity
of a polypeptide selected from the group consisting of SEQ ID
NOs:16 and 18; or a combination thereof.
7. The N. tabacum plant, or part thereof, of claim 5, wherein leaf
from the mutant N. tabacum plant exhibits comparable or better
quality than leaf from the plant lacking said mutation.
8. Cured leaf from the N. tabacum plant of claim 5.
9. A tobacco product comprising the cured leaf of claim 8.
10. The tobacco product of claim 9, consisting of cigarettes,
smokeless tobacco products, tobacco derived nicotine products,
cigarillos, non-ventilated recess filter cigarettes, vented recess
filter cigarettes, cigars, snuff, pipe tobacco, cigar tobacco,
cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco,
and cut tobacco.
11. A nucleic acid molecule comprising a first nucleic acid
sequence between 15 and 500 nucleotides in length and a second
nucleic acid sequence between 15 and 500 nucleotides in length,
wherein the first nucleic acid sequence has a region of
complementarity comprising at least 15 contiguous nucleotides that
are complementary to the second nucleic acid sequence, wherein said
region of complementarity of the first nucleic acid sequence
comprises at least 15 contiguous nucleotides of the sequence shown
in SEQ ID NO: 15 or 17.
12. The nucleic acid molecule of claim 11, further comprising a
spacer nucleic acid sequence between the first nucleic acid
sequence and the second nucleic acid sequence.
13. A method of making a Nicotiana tabacum plant, comprising:
transforming N. tabacum cells with the nucleic acid molecule of
claim 11 to produce transgenic N. tabacum cells; regenerating
transgenic N. tabacum plants from the transgenic N. tabacum cells;
and selecting at least one transgenic N. tabacum plant that
comprises the nucleic acid molecule.
14. The method of claim 13, further comprising identifying at least
one transgenic N. tabacum plant comprising a reduced amount of mRNA
corresponding to a sequence selected from the group consisting of
SEQ ID NOs:15 and 17; a reduced amount of a polypeptide comprising
SEQ ID NO:16 or 18; reduced activity of a polypeptide comprising
SEQ ID NO:16 or 18; a reduced concentration of thylakoid membranes
in the photosystems; a reduced amount of total chlorophyll; an
increased ratio of chlorophyll a to chlorophyll b; increased
biomass relative to a N. tabacum plant not transformed with said
nucleic acid molecule; or a combination thereof.
15. The method of claim 13, wherein leaf from the selected
transgenic N. tabacum plant exhibits comparable or better quality
than leaf from the non-transformed N. tabacum plant.
16. A transgenic Nicotiana tabacum plant comprising a vector, a
vector comprising a nucleic acid sequence having a length of 15 to
500 nucleotides and having at least 95% sequence identity to the
complement of SEQ ID NO: 15 or 17; wherein the expression of an
endogenous gene comprising SEQ ID NO:15 or 17 is reduced relative
to a control plant lacking said vector.
17. The transgenic N. tabacum plant of claim 16, wherein leaf from
the plant exhibits comparable or better quality than leaf from a N.
tabacum plant lacking the nucleic acid molecule.
18. Cured leaf from the transgenic N. tabacum plant of claim
16.
19. A tobacco product comprising the cured leaf of claim 18.
20. The tobacco product of claim 19, selected from the group
consisting of smokeless tobacco products, cigarillos,
non-ventilated recess filter cigarettes, vented recess filter
cigarettes, cigars, snuff, pipe tobacco, cigar tobacco, cigarette
tobacco, chewing tobacco, leaf tobacco, shredded tobacco, and cut
tobacco.
21. The N. tabacum plant, or part thereof, of claim 5, wherein leaf
from a mutant plant comprises a reduced concentration of thylakoid
membranes in the photosystems; a reduced amount of total
chlorophyll; an increased ratio of chlorophyll a to chlorophyll b;
increased biomass relative to leaf from a plant lacking the
mutation; or a combination thereof.
Description
TECHNICAL FIELD
This disclosure generally relates to plants that exhibit altered
photosynthesis.
BACKGROUND
During photosynthesis, at low sunlight intensities, all absorbed
photons are utilized efficiently to drive electrons in the
electron-transport chain. As the level of irradiance increases
further, photosynthesis becomes saturated and reaches a plateau due
to the fact that the carbon reactions cannot keep up with the
linear increase in light absorption. Plant lines with a wild type
light-harvesting antenna system reach this light intensity for
saturation at lower levels of irradiance than their mutant
counterparts. The sunlight harvested by the chlorophyll antenna
exceeds the maximal operational capacity of the electron-transport
chain and of the carbon reactions of photosynthesis, rendering the
excess absorbed photons useless. Under bright sunlight conditions
(2500 .mu.mol photons m-2 s-1), wild type lines with their fully
developed light-harvesting antenna utilize photons inefficiently;
only about 20% of the incoming sunlight energy is converted into
useful photosynthesis, while excess absorbed energy is dissipated
by the non-photochemical quenching (NPQ) process.
SUMMARY
Tobacco plants that exhibit altered photosynthesis are provided
herein, as well as methods of making and using such plants.
In one aspect, a method of making a Nicotiana tabacum plant is
provided. Such a method typically includes inducing mutagenesis in
N. tabacum cells to produce mutagenized N. tabacum cells; obtaining
one or more N. tabacum plants from the mutagenized N. tabacum
cells; and identifying at least one of the N. tabacum plants that
comprises a mutated TLA or CAO sequence. Representative TLA or CAO
sequences have at least 95% sequence identity to a sequence shown
in SEQ ID NO: 11, 13, 15, 17, 19, 21, 23 or 25.
Such a method further can include identifying at least one of the
N. tabacum plants that exhibits reduced amount of TLA or CAO mRNA;
reduced amount of TLA or CAO polypeptide; reduced activity of a TLA
or CAO polypeptide; reduced concentration of thylakoid membranes in
the photosystems; reduced amount of total chlorophyll; increased
ratio of chlorophyll a to chlorophyll b; and/or increased biomass
relative to a N. tabacum plant lacking a mutated TLA or CAO
sequence.
In some embodiments, leaf from the mutant N. tabacum plant exhibits
comparable or better quality than leaf from the plant lacking a
mutated TLA or CAO sequence. In some embodiments, the N. tabacum
plant is a Burley type, a dark type, a flue-cured type, or an
Oriental type.
In another aspect, a variety of Nicotiana tabacum is provided. Such
a variety typically includes plants having a mutation in an
endogenous nucleic acid, where the wild type endogenous nucleic
acid encodes the TLA or CAO sequence shown in SEQ ID NO:12, 14, 16,
18, 20, 22, 24 or 26. Typically, leaf from the mutant plants
exhibits reduced amount of TLA or CAO mRNA; reduced amount of TLA
or CAO polypeptide; reduced activity of a TLA or CAO polypeptide;
reduced concentration of thylakoid membranes in the photosystems;
reduced amount of total chlorophyll; increased ratio of chlorophyll
a to chlorophyll b; and/or increased biomass relative to leaf from
a plant lacking the mutation. In some embodiments, leaf from the
mutant N. tabacum plant exhibits comparable or better quality than
leaf from the plant lacking a mutated TLA sequence.
In another aspect, cured leaf from one of the N. tabacum varieties
described herein is provided. In still another aspect, a tobacco
product that includes such cured leaf is provided. Representative
tobacco products include, without limitation, cigarettes, smokeless
tobacco products, tobacco-derived nicotine products, cigarillos,
non-ventilated recess filter cigarettes, vented recess filter
cigarettes, cigars, snuff, electronic cigarettes, e-vapor products,
pipe tobacco, cigar tobacco, cigarette tobacco, chewing tobacco,
leaf tobacco, shredded tobacco, and cut tobacco.
In still another aspect, a RNA nucleic acid molecule is provided.
Such a RNA nucleic acid molecule typically includes a first nucleic
acid between 15 and 500 nucleotides in length and a second nucleic
acid between 15 and 500 nucleotides in length, where the first
nucleic acid has a region of complementarity to the second nucleic
acid, and where the first nucleic acid comprises at least 15
contiguous nucleotides of the sequence shown in SEQ ID NO: 11, 13,
15, 17, 19, 21, 23 or 25. In some embodiments, the RNA nucleic acid
molecule further includes a spacer nucleic acid between the first
nucleic acid and the second nucleic acid.
In one aspect, a method of making a Nicotiana tabacum plant is
provided. Such a method typically includes transforming N. tabacum
cells with the nucleic acid molecule of claim 12 to produce
transgenic N. tabacum cells; regenerating transgenic N. tabacum
plants from the transgenic N. tabacum cells; and selecting at least
one transgenic N. tabacum plant that comprises the nucleic acid
molecule or the construct. In some embodiments, such a method
further includes identifying at least one transgenic N. tabacum
plant having reduced amount of TLA or CAO mRNA; reduced amount of
TLA or CAO polypeptide; reduced activity of a TLA or CAO
polypeptide; reduced concentration of thylakoid membranes in the
photosystems; reduced amount of total chlorophyll; increased ratio
of chlorophyll a to chlorophyll b; and/or increased biomass
relative to a N. tabacum plant not transformed with the nucleic
acid molecule. In some embodiments, leaf from the selected
transgenic N. tabacum plant exhibits comparable or better quality
than leaf from the non-transformed N. tabacum plant.
In another aspect, a transgenic Nicotiana tabacum plant is provided
that includes a vector, where the vector includes a RNA nucleic
acid molecule having a length of 15 to 500 nucleotides and has at
least 95% sequence identity to a TLA or CAO nucleic acid shown in
SEQ ID NO: 11, 13, 15, 17, 19, 21, 23 or 25. In some embodiments,
leaf from the plant exhibits comparable or better quality than leaf
from a N. tabacum plant lacking the nucleic acid molecule.
In one aspect, cured leaf from such transgenic N. tabacum plants is
provided. In one aspect, tobacco products that include such cured
leaf are provided. Representative tobacco products include, without
limitation, smokeless tobacco products, tobacco-derived nicotine
products, cigarillos, non-ventilated recess filter cigarettes,
vented recess filter cigarettes, cigars, snuff, pipe tobacco, cigar
tobacco, cigarette tobacco, chewing tobacco, leaf tobacco, shredded
tobacco, and cut tobacco.
Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the methods and compositions of
matter belong. Although methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the methods and compositions of matter, suitable methods and
materials are described below. In addition, the materials, methods,
and examples are illustrative only and not intended to be limiting.
All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a photograph of a wild type tobacco plant (left) and a
T0 tobacco plant transgenic for a RNAi nucleic acid molecule
directed toward TLA2 (TLA2i-1; right).
FIG. 1B is a photograph of a T1 tobacco plant transgenic for a RNAi
nucleic acid molecule directed toward TLA2 (TLA2i-1).
FIG. 2A is a photograph of T0 tobacco plants transgenic for a RNAi
nucleic acid molecule directed toward TLA3 (the three plants on the
left, from left to right: TLA3i-5, TLA3i-2, and TLA3i-3) and a wild
type tobacco plant (far right).
FIG. 2B is a photograph of a T1 tobacco plant transgenic for a
nucleic acid molecule directed toward TLA3 (TLA3i-3).
FIG. 2C is a photograph of a T1 tobacco plant transgenic for a
nucleic acid molecule directed toward TLA3 (TLA3i1-1).
FIG. 3 is a photograph of a wild type tobacco plant (left) and a T0
tobacco plant transgenic for a RNAi nucleic acid molecule directed
toward TLA4 (TLA4; right).
FIG. 4A is a graph showing the amount of TLA2 mRNA in T0 tobacco
plants transgenic for RNAi nucleic acid molecules directed toward
TLA2.
FIG. 4B is a graph showing the amount of TLA3 mRNA in T0 tobacco
plants transgenic for RNAi nucleic acid molecules directed toward
TLA3.
FIG. 5A is a photograph of a T0 generation tobacco plant transgenic
for CAOi-1.
FIG. 5B is a photograph of a T0 generation tobacco plant transgenic
for CAOi-2.
DETAILED DESCRIPTION
Photosynthetic organisms (e.g., green plants, algae, and many
bacteria) contain reaction centers, which is a complex of proteins,
pigments and co-factors that perform the photosynthetic conversion
of light to energy via a multitude of electron transfer steps.
Despite the evolutionary distances between such photosynthetic
organisms, the reaction centers possess remarkable homology. In
contrast, the light-harvesting complexes in the various
photosynthetic organisms differ. The current methods used to
measure light absorption and utilization in plants and microalgae
are described in Melis and Thielen (1980, Biochim. Biophys. Acta,
589:275-86) and Melis (1989, Philos. Trans. R. Soc. Lond. B,
323:397-409).
During the process of photosynthesis, the light-harvesting complex,
which typically surrounds the reaction center, absorbs the light
(e.g., sunlight). In plants, the light energy is absorbed by the
light-harvesting antenna complex and is transferred to two
chlorophyll a molecules, which are embedded in the reaction center.
As described herein, light-harvesting antenna complex size can be
inhibited or reduced in tobacco using, for example, mutagenesis or
RNAi, to diminish over-absorption of sunlight at the higher canopy.
Diminishing over-absorption of sunlight in the higher canopy of the
plant can minimize wasteful dissipation of energy, while, at the
same time, allowing for a far greater transmittance of sunlight
deeper into the lower canopy by eliminating unwanted shading,
particularly under high density growth conditions.
Tobacco genes and the encoded proteins were screened to identify
those involved in harvesting light; those sequences identified in
the screen were evaluated further to identify their mode of action.
The sequences identified herein can be inhibited (e.g., by RNA
interference and/or mutation) to result in smaller light-harvesting
chlorophyll antenna size, which ultimately results in a plant that
exhibits substantially improved photosynthetic efficiency. A number
of tobacco sequences (e.g., truncated light-harvesting antenna
(TLA) 2, TLA3, TLA4 and CAO (Chlorophyllide a oxygenase)) as well
as corresponding homologues from Chlamydomonas and/or Arabidopsis
were obtained.
Four TLA-related genes, TLA2 and TLA2 Homo (encoding the CpFTSY
protein), TLA3 and TLA3 Homo (encoding the CpSRP43 protein), and
TLA4 (encoding the CpSRP54 protein) were obtained from Nicotiana
tabacum, as well as three CAO genes (CAO-2, COA-3 and CAO-4). Based
on sequence alignment, CAO-2 appears to have originated from
Nicotiana tometosiformis, while both CAO-3 and COA-4 originated
from Nicotiana sylvestris.
As described in more detail below, the expression of one or more of
the sequences described herein can be inhibited or reduced using,
for example, mutagenesis or inhibitory RNA (RNAi). The resulting
plants can be evaluated for total chlorophyll, as well as the ratio
of chlorophyll a:chlorophyll b and/or the photosynthetic apparatus
size in Photosystem I (PSI) and/or Photosystem II (PSII). Sequences
that, when their expression is knocked down or completely
eliminated, result in a higher ratio of chlorophyll a:b and reduced
antenna size in PSI and/or PSII systems were desired, as it is
these sequences that will substantially improve photosynthetic
efficiency.
As described herein, modification of TLA sequences and CAO
sequences in tobacco results in smaller light-harvesting
chlorophyll antenna complex size by reducing antenna number and a
substantially improved photosynthetic efficiency. The modified
tobacco lines further exhibit enhanced productivity (e.g.,
increased biomass).
Specifically, for example, knocking down TLA2 resulted in plants
that grow slower than wild type plants, plants that have a ratio of
Chlorophyll a:Chlorophyll b similar to wild type, plants that have
an antenna size in PSI that is similar to wild type, plants that
have an antenna size in PSII that is reduced compared to wild type
plants, and plants that have a lighter leaf color than wild type
plants due to the reduction of total chlorophyll content.
In addition, knocking down TLA3 resulted in plants that grow at a
similar rate to wild type plants, plants that have a ratio of
Chlorophyll a:Chlorophyll b that is increased relative to wild type
plants, plants that have an antenna size in both PSI and PSII that
is reduced compared to wild type plants, plants in which the amount
of total chlorophyll increased from low to normal levels during
maturation, relative to wild type plants.
Further, knocking down TLA4 resulted in plants in which the amount
of total chlorophyll was reduced, but both the ratio of chlorophyll
a:chlorophyll b and the antenna size in both PSI and PSII were
unchanged.
Light Harvesting Antenna Nucleic Acids and Polypeptides
Nucleic acids encoding TLA2, TLA2-homo, TLA3, TLA3-homo and TLA4
from N. tabacum are shown in SEQ ID NOs: 11, 13, 15, 17, and 19,
respectively, and nucleic acids encoding CAO2, CAO3 and CAO4 from
N. tabacum are shown in SEQ ID NOs: 21, 23, and 25, respectively.
Unless otherwise specified, nucleic acids referred to herein can
refer to DNA and RNA, and also can refer to nucleic acids that
contain one or more nucleotide analogs or backbone modifications.
Nucleic acids can be single stranded or double stranded, and linear
or circular, both of which usually depend upon the intended
use.
As used herein, an "isolated" nucleic acid molecule is a nucleic
acid molecule that is free of sequences that naturally flank one or
both ends of the nucleic acid in the genome of the organism from
which the isolated nucleic acid molecule is derived (e.g., a cDNA
or genomic DNA fragment produced by PCR or restriction endonuclease
digestion). Such an isolated nucleic acid molecule is generally
introduced into a vector (e.g., a cloning vector, or an expression
vector) for convenience of manipulation or to generate a fusion
nucleic acid molecule, discussed in more detail below. In addition,
an isolated nucleic acid molecule can include an engineered nucleic
acid molecule such as a recombinant or a synthetic nucleic acid
molecule.
The sequence of the TLA2, TLA2-homo, TLA3, TLA3-homo and TLA4
polypeptides from N. tabacum are shown in SEQ ID NOs: 12, 14, 16,
18, and 20, respectively, and the sequences of the CAO2, CAO3 and
CAO4 polypeptides from N. tabacum are shown in SEQ ID NOs: 22, 24,
and 26, respectively. As used herein, a "purified" polypeptide is a
polypeptide that has been separated or purified from cellular
components that naturally accompany it. Typically, the polypeptide
is considered "purified" when it is at least 70% (e.g., at least
75%, 80%, 85%, 90%, 95%, or 99%) by dry weight, free from the
polypeptides and naturally occurring molecules with which it is
naturally associated. Since a polypeptide that is chemically
synthesized is, by nature, separated from the components that
naturally accompany it, a synthetic polypeptide is "purified."
Nucleic acids can be isolated using techniques well known in the
art. For example, nucleic acids can be isolated using any method
including, without limitation, recombinant nucleic acid technology,
and/or the polymerase chain reaction (PCR). General PCR techniques
are described, for example in PCR Primer: A Laboratory Manual,
Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory
Press, 1995. Recombinant nucleic acid techniques include, for
example, restriction enzyme digestion and ligation, which can be
used to isolate a nucleic acid. Isolated nucleic acids also can be
chemically synthesized, either as a single nucleic acid molecule or
as a series of oligonucleotides.
Polypeptides can be purified from natural sources (e.g., a
biological sample) by known methods such as DEAE ion exchange, gel
filtration, and hydroxyapatite chromatography. A polypeptide also
can be purified, for example, by expressing a nucleic acid in an
expression vector. In addition, a purified polypeptide can be
obtained by chemical synthesis. The extent of purity of a
polypeptide can be measured using any appropriate method, e.g.,
column chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
Nucleic acids can be detected using any number of amplification
techniques (see, e.g., PCR Primer: A Laboratory Manual, 1995,
Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.; and U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188) with an appropriate pair of
oligonucleotides (e.g., primers). A number of modifications to the
original PCR have been developed and can be used to detect a
nucleic acid. Nucleic acids also can be detected using
hybridization.
Polypeptides can be detected using antibodies. Techniques for
detecting polypeptides using antibodies include enzyme linked
immunosorbent assays (ELISAs), Western blots, immunoprecipitations
and immunofluorescence. An antibody can be polyclonal or
monoclonal. An antibody having specific binding affinity for a
polypeptide can be generated using methods well known in the art.
The antibody can be attached to a solid support such as a
microtiter plate using methods known in the art. In the presence of
a polypeptide, an antibody-polypeptide complex is formed.
Detection (e.g., of an amplification product, a hybridization
complex, or a polypeptide) is oftentimes accomplished using
detectable labels. The term "label" is intended to encompass the
use of direct labels as well as indirect labels. Detectable labels
include enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials.
A construct, sometimes referred to as a vector, containing a
nucleic acid (e.g., a coding sequence or a RNAi nucleic acid
molecule) is provided. Constructs, including expression constructs
(or expression vectors), are commercially available or can be
produced by recombinant DNA techniques routine in the art. A
construct containing a nucleic acid can have expression elements
operably linked to such a nucleic acid, and further can include
sequences such as those encoding a selectable marker (e.g., an
antibiotic resistance gene). A construct can encode a chimeric or
fusion polypeptide (i.e., a first polypeptide operatively linked to
a second polypeptide). Representative first (or second)
polypeptides are those that can be used in purification of the
other (i.e., second (or first), respectively) polypeptide
including, without limitation, 6.times. His (SEQ ID NO:44) tag or
glutathione S-transferase (GST).
Expression elements include nucleic acid sequences that direct and
regulate expression of nucleic acid coding sequences. One example
of an expression element is a promoter sequence. Expression
elements also can include introns, enhancer sequences, response
elements, or inducible elements that modulate expression of a
nucleic acid. Expression elements can be of bacterial, yeast,
insect, mammalian, or viral origin, and vectors can contain a
combination of elements from different origins. As used herein,
operably linked means that a promoter or other expression
element(s) are positioned in a vector relative to a nucleic acid in
such a way as to direct or regulate expression of the nucleic acid
(e.g., in-frame).
Constructs as described herein can be introduced into a host cell.
Many methods for introducing nucleic acids into host cells, both in
vivo and in vitro, are well known to those skilled in the art and
include, without limitation, electroporation, calcium phosphate
precipitation, polyethylene glycol (PEG) transformation, heat
shock, lipofection, microinjection, and viral-mediated nucleic acid
transfer. As used herein, "host cell" refers to the particular cell
into which the nucleic acid is introduced and also includes the
progeny or potential progeny of such a cell. A host cell can be any
prokaryotic or eukaryotic cell. For example, nucleic acids can be
introduced into bacterial cells such as E. coli, or into insect
cells, yeast or mammalian cells (such as Chinese hamster ovary
cells (CHO) or COS cells). Other suitable host cells are known to
those skilled in the art.
RNA Interfering Nucleic Acids and Constructs Containing Same
RNA interference (RNAi), also called post-transcriptional gene
silencing (PTGS), is a biological process in which RNA molecules
inhibit gene expression, typically by causing the destruction of
specific mRNA molecules. Without being bound by theory, it appears
that, in the presence of an antisense RNA molecule that is
complementary to an expressed message (i.e., a mRNA), the two
strands anneal to generate long double-stranded RNA (dsRNA), which
is digested into short (<30 nucleotide) RNA duplexes, known as
small interfering RNAs (siRNAs), by an enzyme known as Dicer. A
complex of proteins known as the RNA Induced Silencing Complex
(RISC) then unwinds siRNAs, and uses one strand to identify and
thereby anneal to other copies of the original mRNA. RISC cleaves
the mRNA within the complementary sequence, leaving the mRNA
susceptible to further degradation by exonucleases, which
effectively silences expression of the encoding gene.
Several methods have been developed that take advantage of the
endogenous machinery to suppress the expression of a specific
target gene and a number of companies offer RNAi design and
synthesis services (e.g., Life Technologies, Applied Biosystems).
In transgenic plants, the use of RNAi can involve the introduction
of long dsRNA (e.g., greater than 50 bps) or siRNAs (e.g., 12 to 23
bps) that have complementarity to the target gene, both of which
are processed by the endogenous machinery. Alternatively, the use
of RNAi can involve the introduction of a small hairpin RNA
(shRNA); shRNA is a nucleic acid that includes the sequence of the
two desired siRNA strands, sense and antisense, on a single strand,
connected by a "loop" or "spacer" nucleic acid. When the shRNA is
transcribed, the two complementary portions anneal
intra-molecularly to form a "hairpin," which is recognized and
processed by the endogenous machinery.
A RNAi nucleic acid molecule as described herein is complementary
to at least a portion of a target mRNA (e.g., a TLA mRNA, a CAO
mRNA), and typically is referred to as an "antisense strand".
Typically, the antisense strand includes at least 15 contiguous
nucleotides of the DNA sequence (e.g., the nucleic acid sequence
shown in SEQ ID NO:11, 13, 15, 17, 19, 21, 23, or 25); it would be
appreciated that the antisense strand has the "RNA equivalent"
sequence of the DNA (e.g., uracils instead of thymines; ribose
sugars instead of deoxyribose sugars).
A RNAi nucleic acid molecule can be, for example, 15 to 500
nucleotides in length (e.g., 15 to 50, 15 to 45, 15 to 30, 16 to
47, 16 to 38, 16 to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19
to 49, 20 to 60, 20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90,
15 to 100, 15 to 300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17
to 74, 17 to 162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to
400, 20 to 35, 20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325,
20 to 400, 20 to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25
to 250, 25 to 300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35
to 250, 200 to 500, 200 to 400, 250 to 450, 250 to 350, or 300 to
400 nucleotides in length).
In some embodiments, the antisense strand (e.g., a first nucleic
acid) can be accompanied by a "sense strand" (e.g., a second
nucleic acid), which is complementary to the antisense strand. In
the latter case, each nucleic acid (e.g., each of the sense and
antisense strands) can be between 15 and 500 nucleotides in length
(e.g., between 15 to 50, 15 to 45, 15 to 30, 16 to 47, 16 to 38, 16
to 29, 17 to 53, 17 to 44, 17 to 38, 18 to 36, 19 to 49, 20 to 60,
20 to 40, 25 to 75, 25 to 100, 28 to 85, 30 to 90, 15 to 100, 15 to
300, 15 to 450, 16 to 70, 16 to 150, 16 to 275, 17 to 74, 17 to
162, 17 to 305, 18 to 60, 18 to 75, 18 to 250, 18 to 400, 20 to 35,
20 to 60, 20 to 80, 20 to 175, 20 to 225, 20 to 325, 20 to 400, 20
to 475, 25 to 45, 25 to 65, 25 to 100, 25 to 200, 25 to 250, 25 to
300, 25 to 350, 25 to 400, 25 to 450, 30 to 280, 35 to 250, 200 to
500, 200 to 400, 250 to 450, 250 to 350, or 300 to 400 nucleotides
in length).
In some embodiments, a spacer nucleic acid, sometimes referred to
as a loop nucleic acid, can be positioned between the sense strand
and the antisense strand. In some embodiments, the spacer nucleic
acid can be an intron (see, for example, Wesley et al., 2001, The
Plant 1, 27:581-90). In some embodiments, although not required,
the intron can be functional (i.e., in sense orientation; i.e.,
spliceable) (see, for example, Smith et al., 2000, Nature,
407:319-20). A spacer nucleic acid can be between 20 nucleotides
and 1000 nucleotides in length (e.g., 25-800, 25-600, 25-400,
50-750, 50-500, 50-250, 100-700, 100-500, 100-300, 250-700,
300-600, 400-700, 500-800, 600-850, or 700-1000 nucleotides in
length).
In some embodiments, a construct can be produced by operably
linking a promoter that is operable in plant cells; a DNA region,
that, when transcribed, produces an RNA molecule capable of forming
a hairpin structure; and a DNA region involved in transcription
termination and polyadenylation. It would be appreciated that the
hairpin structure has two annealing RNA sequences, where one of the
annealing RNA sequences of the hairpin RNA structure includes a
sense sequence identical to at least 20 consecutive nucleotides of
a TLA or CAO nucleotide sequence, and where the second of the
annealing RNA sequences includes an antisense sequence that is
identical to at least 20 consecutive nucleotides of the complement
of the TLA or CAO nucleotide sequence. In addition, as indicated
herein, the DNA region can include an intron (e.g., a functional
intron). When present, the intron generally is located between the
two annealing RNA sequences in sense orientation such that it is
spliced out by the cellular machinery (e.g., the splicesome). Such
a construct can be introduced into one or more plant cells to
reduce the phenotypic expression of a nucleic acid (e.g., a nucleic
acid sequence that is normally expressed in a plant cell).
In some embodiments, a construct (e.g., an expression construct)
can include an inverted-duplication of a segment of a TLA or CAO
gene, where the inverted-duplication of the TLA or CAO gene segment
includes a nucleotide sequence substantially identical to at least
a portion of the TLA or CAO gene and the complement of the portion
of the TLA or
CAO gene, respectively. It would be appreciated that a single
promoter can be used to drive expression of the
inverted-duplication of the TLA or CAO gene segment, and that the
inverted-duplication typically contains at least one copy of the
portion of the TLA or CAO gene in the sense orientation. Such a
construct can be introduced into one or more plant cells to delay,
inhibit or otherwise reduce the expression of a TLA or CAO gene in
the plant cells.
Representative RNAi nucleic acid molecules directed toward TLA2,
TLA3 and TLA4 are shown in SEQ ID NOs: 27, 28 and 29, respectively,
and a representative RNAi nucleic acid molecule directed toward
CAO2, CAO3 and CAO4 is shown in SEQ ID NO:30. The sense strand and
antisense strand are identified with dashed underlining, and a
spacer or loop sequence lies between. It would be appreciated by
the skilled artisan that the region of complementarity, between the
antisense strand of the RNAi and the mRNA or between the antisense
strand of the RNAi and the sense strand of the RNAi, can be over
the entire length of the RNAi nucleic acid molecule, or the region
of complementarity can be less than the entire length of the RNAi
nucleic acid molecule. For example, a region of complementarity can
refer to, for example, at least 15 nucleotides in length up to, for
example, 500 nucleotides in length (e.g., at least 15, 16, 17, 18,
19, 20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150, 180, 200,
250, 300, 320, 385, 420, 435 nucleotides in length up to, e.g., 30,
35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175, 200, 225,
250, 280, 300, 325, 350, 400, 450, or 475 nucleotides in length).
In some embodiments, a region of complementarity can refer to, for
example, at least 15 contiguous nucleotides in length up to, for
example, 500 contiguous nucleotides in length (e.g., at least 15,
16, 17, 18, 19, 20, 25, 28, 30, 35, 49, 50, 60, 75, 80, 100, 150,
180, 200, 250, 300, 320, 385, 420, 435 nucleotides in length up to,
e.g., 30, 35, 36, 40, 45, 49, 50, 60, 65, 75, 80, 85, 90, 100, 175,
200, 225, 250, 280, 300, 325, 350, 400, 450, or 475 contiguous
nucleotides in length).
It would be appreciated by the skilled artisan that complementary
can refer to, for example, 100% sequence identity between the two
nucleic acids. In addition, however, it also would be appreciated
by the skilled artisan that complementary can refer to, for
example, slightly less than 100% sequence identity (e.g., at least
95%, 96%, 97%, 98%, or 99% sequence identity). In calculating
percent sequence identity, two nucleic acids are aligned and the
number of identical matches of nucleotides (or amino acid residues)
between the two nucleic acids (or polypeptides) is determined. The
number of identical matches is divided by the length of the aligned
region (i.e., the number of aligned nucleotides (or amino acid
residues)) and multiplied by 100 to arrive at a percent sequence
identity value. It will be appreciated that the length of the
aligned region can be a portion of one or both nucleic acids up to
the full-length size of the shortest nucleic acid. It also will be
appreciated that a single nucleic acid can align with more than one
other nucleic acid and hence, can have different percent sequence
identity values over each aligned region.
The alignment of two or more nucleic acids to determine percent
sequence identity can be performed using the computer program
ClustalW and default parameters, which allows alignments of nucleic
acid or polypeptide sequences to be carried out across their entire
length (global alignment). Chenna et al., 2003, Nucleic Acids Res.,
31(13):3497-500. ClustalW calculates the best match between a query
and one or more subject sequences (nucleic acid or polypeptide),
and aligns them so that identities, similarities and differences
can be determined. Gaps of one or more residues can be inserted
into a query sequence, a subject sequence, or both, to maximize
sequence alignments. For fast pairwise alignment of nucleic acid
sequences, the default parameters can be used (i.e., word size: 2;
window size: 4; scoring method: percentage; number of top
diagonals: 4; and gap penalty: 5); for an alignment of multiple
nucleic acid sequences, the following parameters can be used: gap
opening penalty: 10.0; gap extension penalty: 5.0; and weight
transitions: yes. For fast pairwise alignment of polypeptide
sequences, the following parameters can be used: word size: 1;
window size: 5; scoring method: percentage; number of top
diagonals: 5; and gap penalty: 3. For multiple alignment of
polypeptide sequences, the following parameters can be used: weight
matrix: blosum; gap opening penalty: 10.0; gap extension penalty:
0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser,
Asn, Asp, Gln, Glu, Arg, and Lys; and residue-specific gap
penalties: on. ClustalW can be run, for example, at the Baylor
College of Medicine Search Launcher website or at the European
Bioinformatics Institute website on the World Wide Web.
The skilled artisan also would appreciate that complementary can be
dependent upon, for example, the conditions under which two nucleic
acids hybridize. Hybridization between nucleic acids is discussed
in detail in Sambrook et al. (1989, Molecular Cloning: A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Sections 7.37-7.57, 9.47-9.57, 11.7-11.8, and
11.45-11.57). Sambrook et al. disclose suitable Southern blot
conditions for oligonucleotide probes less than about 100
nucleotides (Sections 11.45-11.46). The Tm between a nucleic acid
that is less than 100 nucleotides in length and a second nucleic
acid can be calculated using the formula provided in Section 11.46.
Sambrook et al. additionally disclose Southern blot conditions for
oligonucleotide probes greater than about 100 nucleotides (see
Sections 9.47-9.54). The Tm between a nucleic acid greater than 100
nucleotides in length and a second nucleic acid can be calculated
using the formula provided in Sections 9.50-9.51 of Sambrook et
al.
The conditions under which membranes containing nucleic acids are
prehybridized and hybridized, as well as the conditions under which
membranes containing nucleic acids are washed to remove excess and
non-specifically bound probe, can play a significant role in the
stringency of the hybridization. Such hybridizations and washes can
be performed, where appropriate, under moderate or high stringency
conditions. For example, washing conditions can be made more
stringent by decreasing the salt concentration in the wash
solutions and/or by increasing the temperature at which the washes
are performed. Simply by way of example, high stringency conditions
typically include a wash of the membranes in 0.2.times.SSC at
65.degree. C.
In addition, interpreting the amount of hybridization can be
affected, for example, by the specific activity of the labeled
oligonucleotide probe, by the number of probe-binding sites on the
template nucleic acid to which the probe has hybridized, and by the
amount of exposure of an autoradiograph or other detection medium.
It will be readily appreciated by those of ordinary skill in the
art that although any number of hybridization and washing
conditions can be used to examine hybridization of a probe nucleic
acid molecule to immobilized target nucleic acids, it is more
important to examine hybridization of a probe to target nucleic
acids under identical hybridization, washing, and exposure
conditions. Preferably, the target nucleic acids are on the same
membrane. A nucleic acid molecule is deemed to hybridize to a
nucleic acid, but not to another nucleic acid, if hybridization to
a nucleic acid is at least 5-fold (e.g., at least 6-fold, 7-fold,
8-fold, 9-fold, 10-fold, 20-fold, 50-fold, or 100-fold) greater
than hybridization to another nucleic acid. The amount of
hybridization can be quantified directly on a membrane or from an
autoradiograph using, for example, a PhosphorImager or a
Densitometer (Molecular Dynamics, Sunnyvale, Calif.).
A construct (also known as a vector) containing a RNAi nucleic acid
molecule is provided. Constructs, including expression constructs,
are described herein and are known to those of skill in the art.
Expression elements (e.g., promoters) that can be used to drive
expression of a RNAi nucleic acid molecule are known in the art and
include, without limitation, constitutive promoters such as,
without limitation, the cassava mosaic virus (CsMVM) promoter, the
cauliflower mosaic virus (CaMV) 35S promoter, the actin promoter,
or the glyceraldehyde-3-phosphate dehydrogenase promoter, or
tissue-specific promoters such as, without limitation,
root-specific promoters such as the putrescine N-methyl transferase
(PMT) promoter or the quinolinate phosphosibosyltransferase (QPT)
promoter. It would be understood by a skilled artisan that a sense
strand and an antisense strand can be delivered to and expressed in
a target cell on separate constructs, or the sense and antisense
strands can be delivered to and expressed in a target cell on a
single construct (e.g., in one transcript). As discussed herein, a
RNAi nucleic acid molecule delivered and expressed on a single
strand also can include a spacer nucleic acid (e.g., a loop nucleic
acid) such that the RNAi forms a small hairpin (shRNA).
Transgenic Plants and Methods of Making Transgenic Plants
Transgenic N. tabacum plants are provided that contain a transgene
encoding at least one RNAi molecule, which, when transcribed,
silences expression of any of the TLA or CAO sequences described
herein. As used herein, silencing can refer to complete elimination
or essentially complete elimination of the TLA or CAO mRNA,
resulting in 100% or essentially 100% reduction (e.g., greater than
95% reduction; e.g., greater than 96%, 97%, 98% or 99% reduction)
in the amount of encoded TLA or CAO polypeptide; silencing also can
refer to partial elimination of the TLA or CAO mRNA (e.g.,
eliminating about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%
or more of the TLA or CAO mRNA), resulting in a reduction (e.g.,
about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, but
not complete elimination) in the amount of the encoded TLA or CAO
polypeptide.
A RNAi nucleic acid molecule can be transcribed using a plant
expression vector. Methods of introducing a nucleic acid (e.g., a
heterologous nucleic acid) into plant cells (e.g., N. tabacum
cells) are known in the art and include, for example, particle
bombardment, Agrobacterium-mediated transformation, microinjection,
polyethylene glycol-mediated transformation (e.g., of protoplasts,
see, for example, Yoo et al. (2007, Nature Protocols,
2(7):1565-72)), liposome-mediated DNA uptake, or
electroporation.
Following transformation, the transgenic plant cells can be
regenerated into transgenic tobacco plants. The regenerated
transgenic plants can be screened for the presence of the transgene
(e.g., a RNAi nucleic acid molecule) and/or one or more of the
resulting phenotypes (e.g., reduced amount of TLA or CAO mRNA;
reduced amount of TLA or CAO polypeptide; reduced activity of a TLA
or CAO polypeptide; reduced concentration of thylakoid membranes in
the photosystems; reduced amount of total chlorophyll; increased
ratio of chlorophyll a to chlorophyll b; and/or increased
biomass).
Methods of detecting alkaloids (e.g., nicotine) or TSNAs, and
methods of determining the amount of one or more alkaloids or TSNAs
are known in the art. For example, high performance liquid
chromatography (HPLC)-mass spectroscopy (MS) (HPLC-MS) or high
performance thin layer chromatography (HPTLC) can be used to detect
the presence of one or more alkaloids and/or determine the amount
of one or more alkaloids. In addition, any number of chromatography
methods (e.g., gas chromatography/thermal energy analysis (GC/TEA),
liquid chromatography/mass spectrometry (LC/MS), and ion
chromatography (IC)) can be used to detect the presence of one or
more TSNAs and/or determine the amount of one or more TSNAs.
As used herein, "reduced" or "reduction" refers to a decrease
(e.g., a statistically significant decrease), in green leaf or
cured leaf, of/in one or more of the following: a) the amount of
TLA or CAO mRNA; b) the amount of TLA or CAO polypeptide; c) the
activity of a TLA or CAO polypeptide; d) the concentration of
thylakoid membranes in the photosystems measured
spectrophotometrically from the amplitude of the light-minus-dark
absorbance difference signal at 800 nm (P800) for PSI and 320 nm
(QA) for PSII (see, for example, Melis & Brown, 1980, PNAS USA,
77(8):4712-6; and Melis, 1989, Philos. Trans. R. Soc. Lond. B,
323:397-409); and/or e) the amount of total chlorophyll. As used
herein, "reduced" or "reduction" refers to a decrease in any of the
above by at least about 5% up to about 95% (e.g., about 5% to about
10%, about 5% to about 20%, about 5% to about 50%, about 5% to
about 75%, about 10% to about 25%, about 10% to about 50%, about
10% to about 90%, about 20% to about 40%, about 20% to about 60%,
about 20% to about 80%, about 25% to about 75%, about 50% to about
75%, about 50% to about 85%, about 50% to about 95%, and about 75%
to about 95%) relative to similarly-treated leaf (e.g., green or
cured) from a tobacco plant lacking the transgene. As used herein,
statistical significance refers to a p-value of less than 0.05,
e.g., a p-value of less than 0.025 or a p-value of less than 0.01,
using an appropriate measure of statistical significance, e.g., a
one-tailed two sample t-test.
As used herein, "increased" refers to an increase (e.g., a
statistically significant increase), in green leaf or cured leaf,
of the ratio of chlorophyll a/chlorophyll b or in plant biomass. As
used herein, "increased" refers to an increase in any of the above
by at least about 5% up to about 95% (e.g., about 5% to about 10%,
about 5% to about 20%, about 5% to about 50%, about 5% to about
75%, about 10% to about 25%, about 10% to about 50%, about 10% to
about 90%, about 20% to about 40%, about 20% to about 60%, about
20% to about 80%, about 25% to about 75%, about 50% to about 75%,
about 50% to about 85%, about 50% to about 95%, and about 75% to
about 95%) relative to similarly-treated leaf (e.g., green or
cured) from a tobacco plant lacking the transgene. As used herein,
statistical significance refers to a p-value of less than 0.05,
e.g., a p-value of less than 0.025 or a p-value of less than 0.01,
using an appropriate measure of statistical significance, e.g., a
one-tailed two sample t-test.
Leaf from progeny plants also can be screened for the presence of
the transgene and/or the resulting phenotype, and plants exhibiting
the desired phenotype can be selected. As described herein, leaf
from such transgenic plants can exhibit reduced amount of TLA or
CAO mRNA; reduced amount of TLA or CAO polypeptide; reduced
activity of a TLA or CAO polypeptide; reduced concentration of
thylakoid membranes in the photosystems; reduced amount of total
chlorophyll; increased ratio of chlorophyll a to chlorophyll b;
and/or increased biomass in the plant (e.g., compared to leaf from
a plant lacking or not transcribing the RNAi). Leaf from
regenerated transgenic plants can be screened (e.g., reduced amount
of TLA or CAO mRNA; reduced amount of TLA or CAO polypeptide;
reduced activity of a TLA or CAO polypeptide; reduced concentration
of thylakoid membranes in the photosystems; reduced amount of total
chlorophyll; increased ratio of chlorophyll a to chlorophyll b;
and/or increased biomass), and the desired plants (e.g., having
leaf that exhibit reduced amount of TLA or CAO mRNA; reduced amount
of TLA or CAO polypeptide; reduced activity of a TLA or CAO
polypeptide; reduced concentration of thylakoid membranes in the
photosystems; reduced amount of total chlorophyll; increased ratio
of chlorophyll a to chlorophyll b; and/or increased biomass),
compared to the amount in a leaf from a corresponding
non-transgenic plant, can be selected and, for example, used in a
breeding program.
Transgenic plants exhibiting the desired phenotype can be used, for
example, in a breeding program. Breeding is carried out using known
procedures. Successful crosses yield Fi plants that are fertile and
that can be backcrossed with one of the parents if desired. In some
embodiments, a plant population in the F2 generation is screened
for the presence of a transgene and/or the resulting phenotype
using standard methods (e.g., amplification, hybridization and/or
chemical analysis of the leaf). Selected plants are then crossed
with one of the parents and the first backcross (BC1) generation
plants are self-pollinated to produce a BC1F2 population that is
again screened. The process of backcrossing, self-pollination, and
screening is repeated, for example, at least four times until the
final screening produces a plant that is fertile and reasonably
similar to the recurrent parent. This plant, if desired, is
self-pollinated and the progeny are subsequently screened again to
confirm that the plant contains the transgene and exhibits variant
gene expression. Breeder's seed of the selected plant can be
produced using standard methods including, for example, field
testing and/or chemical analyses of leaf (e.g., cured leaf).
The result of a plant breeding program using the transgenic tobacco
plants described herein are novel and useful varieties, lines, and
hybrids. As used herein, the term "variety" refers to a population
of plants that share constant characteristics which separate them
from other plants of the same species. A variety is often, although
not always, sold commercially. While possessing one or more
distinctive traits, a variety is further characterized by a very
small overall variation between individual with that variety. A
"pure line" variety may be created by several generations of
self-pollination and selection, or vegetative propagation from a
single parent using tissue or cell culture techniques. A "line," as
distinguished from a variety, most often denotes a group of plants
used non-commercially, for example, in plant research. A line
typically displays little overall variation between individuals for
one or more traits of interest, although there may be some
variation between individuals for other traits.
A variety can be essentially derived from another line or variety.
As defined by the International Convention for the Protection of
New Varieties of Plants (Dec. 2, 1961, as revised at Geneva on Nov.
10, 1972, On Oct. 23, 1978, and on Mar. 19, 1991), a variety is
"essentially derived" from an initial variety if: a) it is
predominantly derived from the initial variety, or from a variety
that is predominantly derived from the initial variety, while
retaining the expression of the essential characteristics that
result from the genotype or combination of genotypes of the initial
variety; b) it is clearly distinguishable from the initial variety;
and c) except for the differences which result from the act of
derivation, it conforms to the initial variety in the expression of
the essential characteristics that result from the genotype or
combination of genotypes of the initial variety. Essentially
derived varieties can be obtained, for example, by the selection of
a natural or induced mutant, a somaclonal variant, a variant
individual plant from the initial variety, backcrossing, or
transformation.
Hybrid tobacco varieties can be produced by preventing
self-pollination of female parent plants (i.e., seed parents) of a
first variety, permitting pollen from male parent plants of a
second variety to fertilize the female parent plants, and allowing
Fi hybrid seeds to form on the female plants. Self-pollination of
female plants can be prevented by emasculating the flowers at an
early stage of flower development. Alternatively, pollen formation
can be prevented on the female parent plants using a form of male
sterility. For example, male sterility can be produced by
cytoplasmic male sterility (CMS), nuclear male sterility, genetic
male sterility, molecular male sterility where a transgene inhibits
microsporogenesis and/or pollen formation, or self-incompatibility.
Female parent plants having CMS are particularly useful. In
embodiments in which the female parent plants are CMS, the male
parent plants typically contain a fertility restorer gene to ensure
that the Fi hybrids are fertile. In other embodiments in which the
female parents are CMS, male parents can be used that do not
contain a fertility restorer. Fi hybrids produced from such parents
are male sterile. Male sterile hybrid seed can be interplanted with
male fertile seed to provide pollen for seed-set on the resulting
male sterile plants.
Varieties and lines described herein can be used to form
single-cross tobacco Fi hybrids. In such embodiments, the plants of
the parent varieties can be grown as substantially homogeneous
adjoining populations to facilitate natural cross-pollination from
the male parent plants to the female parent plants. The F2 seed
formed on the female parent plants is selectively harvested by
conventional means. One also can grow the two parent plant
varieties in bulk and harvest a blend of Fi hybrid seed formed on
the female parent and seed formed upon the male parent as the
result of self-pollination. Alternatively, three-way crosses can be
carried out wherein a single-cross Fi hybrid is used as a female
parent and is crossed with a different male parent. As another
alternative, double-cross hybrids can be created wherein the Fi
progeny of two different single-crosses are themselves crossed.
Self-incompatibility can be used to particular advantage to prevent
self-pollination of female parents when forming a double-cross
hybrid.
The tobacco plants used in the methods described herein can
include, but are not limited to, a Burley type, a dark type, a
flue-cured type, or an Oriental type. The tobacco plants used in
the methods described herein typically are from N. tabacum, and can
be from any number of N. tabacum varieties. A variety can be BU 64,
CC 101, CC 200, CC 13, CC 27, CC 33, CC 35,CC 37, CC 65, CC 67, CC
301, CC 400, CC 500, CC 600, CC 700, CC 800, CC 900, CC 1063, Coker
176, Coker 319, Coker 371 Gold, Coker 48, CU 263, DF911, Galpao
tobacco, GL 26H, GL 338, GL 350, GL 395, GL 600, GL 737, GL 939, GL
973, GF 157, GF 318, RJR 901, HB 04P, K 149, K 326, K 346, K 358,
K394, K 399, K 730, NC 196, NC 37NF, NC 471, NC 55, NC 92, NC2326,
NC 95, NC 925, PVH 1118, PVH 1452, PVH 2110, PVH 2254, PVH 2275, VA
116, VA 119, KDH 959, KT 200, KT204LC, KY 10, KY 14, KY 160, KY 17,
KY 171, KY 907, KY907LC, KTY14.times.L8 LC, Little Crittenden,
McNair 373, McNair 944, msKY 14.times.L8, Narrow Leaf Madole, NC
100, NC 102, NC 2000, NC 291, NC 297, NC 299, NC 3, NC 4, NC 5, NC
6, NC7, NC 606, NC 71, NC 72, NC 810, NC BH 129, NC 2002, Neal
Smith Madole, OXFORD 207, Perique tobacco, PVH03, PVH09, PVH19,
PVH50, PVH51, R 610, R 630, R 7-11, R 7-12, RG 17, RG 81, RG H51,
RGH 4, RGH 51, RS 1410, Speight 168, Speight 172, Speight 179,
Speight 210, Speight 220, Speight 225, Speight 227, Speight 234,
Speight G-28, Speight G-70, Speight H-6, Speight H20, Speight NF3,
TI 1406, TI 1269, TN 86, TN86LC, TN 90, TN90LC, TN 97, TN97LC, TN
D94, TN D950, TR (Tom Rosson) Madole, VA 309, or VA359.
Mutant Plants and Methods of Making
Methods of making a N. tabacum plant having a mutation are known in
the art. Mutations can be random mutations or targeted mutations.
For random mutagenesis, cells (e.g., N. tabacum cells) typically
are mutagenized using, for example, a chemical mutagen or ionizing
radiation. Representative chemical mutagens include, without
limitation, nitrous acid, sodium azide, acridine orange, ethidium
bromide, and ethyl methane sulfonate (EMS), while representative
ionizing radiation includes, without limitation, x-rays, gamma
rays, fast neutron irradiation, and UV irradiation. The dosage of
the mutagenic chemical or radiation is determined experimentally
for each type of plant tissue such that a mutation frequency is
obtained that is below a threshold level characterized by lethality
or reproductive sterility. The number of M.sub.1 generation seed or
the size of M.sub.1 plant populations resulting from the mutagenic
treatments are estimated based on the expected frequency of
mutations. For targeted mutagenesis, representative technologies
include TALEN (see, for example, Li et al., 2011, Nucleic Acids
Res., 39(14):6315-25) or zinc-finger (see, for example, Wright et
al., 2005, The Plant 1, 44:693-705). Whether random or targeted, a
mutation can be a point mutation, an insertion, a deletion, a
substitution, or combinations thereof, which are discussed in more
detail below.
The resultant variety of Nicotiana tabacum includes plants having a
mutation in an endogenous TLA nucleic acid (e.g., SEQ ID NOs: 11,
13, 15, 17 or 19) encoding a TLA polypeptide sequence (e.g., SEQ ID
NOs: 12, 14, 16, 18 or 20) or in an endogenous CAO gene (e.g., SEQ
ID NOs: 21, 23 or 25) encoding a CAO polypeptide sequence (e.g.,
SEQ ID NOs: 22, 24 or 26). A mutation in a TLA or CAO sequence as
described herein typically results in reduced expression or
activity of TLA or CAO, which, in turn, results in one or more of
the phenotypes described herein (e.g., reduced concentration of
thylakoid membranes in the photosystems; reduced amount of total
chlorophyll; increased ratio of chlorophyll a to chlorophyll b;
and/or increased biomass), or combinations thereof depending on the
particular combination of sequences that are mutated or otherwise
knocked-down, in the leaf of a mutant plant relative to a plant
lacking the mutation.
As discussed herein, one or more nucleotides can be mutated to
alter the expression and/or function of the encoded polypeptide,
relative to the expression and/or function of the corresponding
wild type polypeptide. It will be appreciated, for example, that a
mutation in one or more of the highly conserved regions would
likely alter polypeptide function, while a mutation outside of
those highly conserved regions would likely have little to no
effect on polypeptide function. In addition, a mutation in a single
nucleotide can create a stop codon, which would result in a
truncated polypeptide and, depending on the extent of truncation,
loss of function.
Suitable types of mutations in a TLA or CAO coding sequence
include, without limitation, insertions of nucleotides, deletions
of nucleotides, or transitions or transversions relative to the
wild-type TLA or CAO coding sequence, respectively. Mutations in
the coding sequence can result in insertions of one or more amino
acids, deletions of one or more amino acids, conservative or
non-conservative amino acid substitutions in the encoded
polypeptide, or truncation of the protein (e.g., by introduction of
a stop codon). In some cases, the coding sequence of a TLA
comprises more than one mutation and/or more than one type of
mutation.
Insertion or deletion of amino acids in a coding sequence, for
example, can disrupt the conformation of the encoded polypeptide.
Amino acid insertions or deletions also can disrupt sites important
for recognition of binding ligand(s) or substrate(s) or for
activity of the polypeptide. It is known in the art that the
insertion or deletion of a larger number of contiguous amino acids
is more likely to render the gene product non-functional, compared
to a smaller number of inserted or deleted amino acids. In
addition, one or more mutations (e.g., a point mutation) can change
the localization of the TLA or CAO polypeptide, introduce a stop
codon to produce a truncated polypeptide, or disrupt an active site
or domain (e.g., a catalytic site or domain, a binding site or
domain) within the polypeptide.
A "conservative amino acid substitution" is one in which one amino
acid residue is replaced with a different amino acid residue having
a similar side chain (see, for example, Dayhoff et al. (1978, in
Atlas of Protein Sequence and Structure, 5 (Suppl. 3):345-352),
which provides frequency tables for amino acid substitutions), and
a non-conservative substitution is one in which an amino acid
residue is replaced with an amino acid residue that does not have a
similar side chain. Non-conservative amino acid substitutions can
replace an amino acid of one class with an amino acid of a
different class. Non-conservative substitutions can make a
substantial change in the charge or hydrophobicity of the gene
product. Non-conservative amino acid substitutions can also make a
substantial change in the bulk of the residue side chain, e.g.,
substituting an alanine residue for an isoleucine residue. Examples
of non-conservative substitutions include a basic amino acid for a
non-polar amino acid, or a polar amino acid for an acidic amino
acid.
Following mutagenesis, M.sub.0 plants are regenerated from the
mutagenized cells and those plants, or a subsequent generation of
that population (e.g., M.sub.1, M.sub.2, M.sub.3, etc.), can be
screened for those carrying a mutation in a TLA of CAO sequence.
Screening for plants carrying a mutation in a TLA of CAO nucleic
acid or polypeptide can be performed directly using methods routine
in the art (e.g., hybridization, amplification, nucleic acid
sequencing, peptide sequencing, combinations thereof) or by
evaluating the phenotype (e.g., reduced amount of TLA or CAO mRNA;
reduced amount of TLA or CAO polypeptide; reduced activity of a TLA
or CAO polypeptide; reduced concentration of thylakoid membranes in
the photosystems; reduced amount of total chlorophyll; increased
ratio of chlorophyll a to chlorophyll b; and/or increased biomass).
It would be understood that the phenotype of a mutant plant (e.g.,
reduced amount of TLA or CAO mRNA; reduced amount of TLA or CAO
polypeptide; reduced activity of a TLA or CAO polypeptide; reduced
concentration of thylakoid membranes in the photosystems; reduced
amount of total chlorophyll; increased ratio of chlorophyll a to
chlorophyll b; and/or increased biomass) would be compared to a
corresponding plant (e.g., having the same varietal background)
that lacks the mutation.
An M.sub.1 tobacco plant may be heterozygous for a mutant allele
and exhibit a wild type phenotype. In such cases, at least a
portion of the first generation of self-pollinated progeny of such
a plant exhibits a wild type phenotype. Alternatively, an M.sub.1
tobacco plant may have a mutant allele and exhibit a mutant
phenotype (e.g., reduced amount of TLA or CAO mRNA; reduced amount
of TLA or CAO polypeptide; reduced activity of a TLA or CAO
polypeptide; reduced concentration of thylakoid membranes in the
photosystems; reduced amount of total chlorophyll; increased ratio
of chlorophyll a to chlorophyll b; and/or increased biomass). Such
plants may be heterozygous and exhibit a mutant phenotype due to a
phenomenon such as dominant negative suppression, despite the
presence of the wild type allele, or such plants may be homozygous
due to independently induced mutations in both alleles.
As used herein, "reduced" or "reduction" refers to a decrease
(e.g., a statistically significant decrease), in green leaf or
cured leaf, of/in one or more of the following: a) the amount of
TLA or CAO mRNA; b) the amount of TLA or CAO polypeptide; c) the
activity of a TLA or CAO polypeptide; d) the concentration of
thylakoid membranes in the photosystems measured
spectrophotometrically from the amplitude of the light-minus-dark
absorbance difference signal at 800 nm (P800) for PSI and 320 nm
(QA) for PSII (see, for example, Melis & Brown, 1980, PNAS USA,
77(8):4712-6; and Melis, 1989, Philos. Trans. R. Soc. Lond. B,
323:397-409); and/or e) the amount of total chlorophyll. As used
herein, "reduced" or "reduction" refers to a decrease in any of the
above by at least about 5% up to about 95% (e.g., about 5% to about
10%, about 5% to about 20%, about 5% to about 50%, about 5% to
about 75%, about 10% to about 25%, about 10% to about 50%, about
10% to about 90%, about 20% to about 40%, about 20% to about 60%,
about 20% to about 80%, about 25% to about 75%, about 50% to about
75%, about 50% to about 85%, about 50% to about 95%, and about 75%
to about 95%) relative to similarly-treated leaf (e.g., green or
cured) from a tobacco plant lacking the transgene. As used herein,
statistical significance refers to a p-value of less than 0.05,
e.g., a p-value of less than 0.025 or a p-value of less than 0.01,
using an appropriate measure of statistical significance, e.g., a
one-tailed two sample t-test.
As used herein, "increased" refers to an increase (e.g., a
statistically significant increase), in green leaf or cured leaf,
of the ratio of chlorophyll a/chlorophyll b or in plant biomass. As
used herein, "increased" refers to an increase in any of the above
by at least about 5% up to about 95% (e.g., about 5% to about 10%,
about 5% to about 20%, about 5% to about 50%, about 5% to about
75%, about 10% to about 25%, about 10% to about 50%, about 10% to
about 90%, about 20% to about 40%, about 20% to about 60%, about
20% to about 80%, about 25% to about 75%, about 50% to about 75%,
about 50% to about 85%, about 50% to about 95%, and about 75% to
about 95%) relative to similarly-treated leaf (e.g., green or
cured) from a tobacco plant lacking the transgene. As used herein,
statistical significance refers to a p-value of less than 0.05,
e.g., a p-value of less than 0.025 or a p-value of less than 0.01,
using an appropriate measure of statistical significance, e.g., a
one-tailed two sample t-test.
A tobacco plant carrying a mutant allele can be used in a plant
breeding program to create novel and useful lines, varieties and
hybrids. Desired plants that possess the mutation can be
backcrossed or self-pollinated to create a second population to be
screened. Backcrossing or other breeding procedures can be repeated
until the desired phenotype of the recurrent parent is recovered.
DNA fingerprinting, SNP or similar technologies may be used in a
marker-assisted selection (MAS) breeding program to transfer or
breed mutant alleles into other tobaccos, as described herein.
In some embodiments, an M.sub.1, M.sub.2, M.sub.3 or later
generation tobacco plant containing at least one mutation is
crossed with a second Nicotiana tabacum plant, and progeny of the
cross are identified in which the mutation(s) is present. It will
be appreciated that the second Nicotiana tabacum plant can be one
of the species and varieties described herein. It will also be
appreciated that the second Nicotiana tabacum plant can contain the
same mutation as the plant to which it is crossed, a different
mutation, or be wild type at the locus. Additionally or
alternatively, a second tobacco line can exhibit a phenotypic trait
such as, for example, disease resistance, high yield, high grade
index, curability, curing quality, mechanical harvesting, holding
ability, leaf quality, height, plant maturation (e.g., early
maturing, early to medium maturing, medium maturing, medium to late
maturing, or late maturing), stalk size (e.g., small, medium, or
large), and/or leaf number per plant (e.g., a small (e.g., 5-10
leaves), medium (e.g., 11-15 leaves), or large (e.g., 16-21) number
of leaves).
Cured Tobacco and Tobacco Products
The methods described herein allow for increasing tobacco biomass
while still maintaining high leaf quality. As described herein,
such methods can include the production of transgenic (using, e.g.,
RNAi or overexpression) or mutant (e.g., random or targeted)
plants.
Leaf quality can be determined, for example, using an Official
Standard Grade published by the Agricultural Marketing Service of
the US Department of Agriculture (7 U.S.C. .sctn. 511); Legacy
Tobacco Document Library (Bates Document #523267826/7833, Jul. 1,
1988, Memorandum on the Proposed Burley Tobacco Grade Index); and
Miller et al., 1990, Tobacco Intern., 192:55-7. For dark-fired
tobacco, leaves typically are obtained from stalk position C, and
the average grade index determined based on Federal Grade and 2004
Price Support for Type 23 Western dark-fired tobacco.
Leaf from the tobacco described herein can be cured, aged,
conditioned, and/or fermented. Methods of curing tobacco are well
known and include, for example, air curing, fire curing, flue
curing and sun curing. Aging also is known and is typically carried
out in a wooden drum (e.g., a hogshead) or cardboard cartons in
compressed conditions for several years (e.g., 2 to 5 years), at a
moisture content of from about 10% to about 25% (see, for example,
U.S. Pat. Nos. 4,516,590 and 5,372,149). Conditioning includes, for
example, a heating, sweating or pasteurization step as described in
US 2004/0118422 or US 2005/0178398, while fermenting typically is
characterized by high initial moisture content, heat generation,
and a 10 to 20% loss of dry weight. See, e.g., U.S. Pat. Nos.
4,528,993; 4,660,577; 4,848,373; and 5,372,149. The tobacco also
can be further processed (e.g., cut, expanded, blended, milled or
comminuted), if desired, and used in a tobacco product.
Tobacco products are known in the art and include any product made
or derived from tobacco that is intended for human consumption,
including any component, part, or accessory of a tobacco product.
Representative tobacco products include, without limitation,
cigarettes, smokeless tobacco products, tobacco-derived nicotine
products, cigarillos, non-ventilated recess filter cigarettes,
vented recess filter cigarettes, cigars, snuff, electronic
cigarettes, e-vapor products, pipe tobacco, cigar tobacco,
cigarette tobacco, chewing tobacco, leaf tobacco, shredded tobacco,
and cut tobacco. Representative smokeless tobacco products include,
for example, chewing tobacco, snus, pouches, films, tablets,
sticks, rods, and the like. Representative cigarettes and other
smoking articles include, for example, smoking articles that
include filter elements or rod elements, where the rod element of a
smokeable material can include cured tobacco within a tobacco
blend. In addition to the reduced-nicotine or reduced-TSNA tobacco
described herein, tobacco products also can include other
ingredients such as, without limitation, binders, plasticizers,
stabilizers, and/or flavorings. See, for example, US 2005/0244521,
US 2006/0191548, US 2012/0024301, US 2012/0031414, and US
2012/0031416 for examples of tobacco products.
In accordance with the present invention, there may be employed
conventional molecular biology, microbiology, biochemical, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. The invention
will be further described in the following examples, which do not
limit the scope of the methods and compositions of matter described
in the claims.
EXAMPLES
Example 1--Sampling, RNA Preparation and Sequencing
Tobacco leaf sample from a burley variety, TN90, was collected. RNA
from the sample was isolated using RNeasy Plant Mini Kit (Qiagen;
MA) and its quality tested using Agilent Plant RNA Nano Kit and a
2100 Bioanalyzer (Agilent Technologies, CA, USA). A cDNA library
was constructed and indexed using a TrueSeq RNA Library Prep Kit v.
2 (Illumina). cDNAs were run on an Illumina HiSeq 2000 under
conditions for 100 bp single reads and a minimum of 30 million
reads per sample. Leaf gene expression in TN90 tobacco was
determined by RNA deep sequencing performed by ArrayXpress
(Raleigh, N.C.).
Example 2--Tobacco TLA2, TLA3 and TLA4 Homologous Gene
Identification, Full Length Gene Cloning
TLA2, TLA3 and TLA4 gene sequences from Chlamydomonas and
Arabidopsis were used to Blast the TN90 burley genomic sequence
data base and leaf RNA sequence data. Five candidate genes were
identified: TLA2, TLA2 Homo, TLA3, TLA3 Homo, and TLA4, and primers
were designed to clone the five candidate genes. Leaf tissue was
collected and a cDNA library was created using the In-Fusion.RTM.
SMARTer.RTM. Directional cDNA Library Construction Kit from
Clontech. Full length candidate genes were amplified using the gene
specific primers designed from predicted full length cDNA
sequences. The full length coding sequences were identified, cloned
and confirmed by sequencing.
TABLE-US-00001 TABLE 1 Primers for TLA gene cloning SEQ ID
Designation SEQUENCE (5' to 3') NO TLA-2-F
ATGGCTTCTCTATTATCTTCTCGTC 1 TLA-2-R TCATGGGAAGATAGCATTAACAAA 2
TLA-2-Homo-F ATGGCTTCTCTATTATCTTCTCGTCTCC 3 TLA-2-Homo-R
TTAAATAGAATCTGCCACATAAATG 4 TLA-3-F ATGGATGCTCTCTTCGTCAATTCCTC 5
TLA-3-R GCCCTTGTTCAACTTACTACATT 6 TLA-3-Homo-F
ATGGATGCTCTGTTCGTCAATTCCT 7 TLA-3-Homo-R TCAACTTACTACATTCTGTTGACCC
8 TLA-4-F ATGGAAGCCACTGCTTCTTTCTCCTCA 9 TLA-4-R
TTAGTTCTTGGCCCCAAAACCACGAG 10
The sequence of the candidate genes are provided as indicated in
Table 2.
TABLE-US-00002 TABLE 2 Candidate genes Gene name Nucleic Acid
Polypeptide TLA2 SEQ ID NO: 11 SEQ ID NO: 12 TLA2-Homo SEQ ID NO:
13 SEQ ID NO: 14 TLA3 SEQ ID NO: 15 SEQ ID NO: 16 TLA3-homo SEQ ID
NO: 17 SEQ ID NO: 18 TLA4 SEQ ID NO: 19 SEQ ID NO: 20 CAO-2 SEQ ID
NO: 21 SEQ ID NO: 22 CAO-3 SEQ ID NO: 23 SEQ ID NO: 24 CAO-4 SEQ ID
NO: 25 SEQ ID NO: 26
Example 3--RNAi Plasmid Construction and Agrobacterium
Transformation
In order to investigate the function of the candidate genes, RNAi
constructs were produced against TLA2, TLA3 and TLA4, and
transgenic plant lines were generated. An Agrobacterium expression
vector (SEQ ID NO:31) was used, which has a CsVMV promoter and a
NOS terminator, as well as a cassette having a kanamycin selection
marker (NPT II) under direction of the actin2 promoter and a NOS
terminator. The nucleic acid constructs carrying each RNAi
construct were introduced into tobacco leaf disc using an
Agrobacterium transformation approach. See, for example, Mayo et
al., 2006, Nat Protoc., 1(3):1105-11; and Horsch et al., 1985,
Science, 227:1229-31.
Briefly, ascetical tobacco plants (Narrow Leaf Madole (NLM)) were
grown in magenta boxes, and leaf discs were cut onto 15.times.150
mm plates. Agrobacterium tumefaciens containing each nucleic acid
construct were collected by centrifugation of 20 ml cell suspension
in 50 ml centrifuge tube at 3500 rpm for 10 minutes. Supernatant
was removed and the Agrobacterium cell pellet was re-suspended in
40 ml liquid re-suspension medium. About 25 ml of the solution was
transferred to each 15.times.100 mm petri plates. In those
15.times.150 mm plates, tobacco leaves were cut into 0.6 cm discs
(avoiding the midrib).
Leaves were placed upside down, a thin layer of MS/B5 liquid
re-suspension medium was added, and leaf discs were produced using
a #15 razor blade. The leaf discs were poked uniformly with a fine
point needle. Eight discs were placed in each regeneration plate
(15.times.100 mm). Agrobacterium tumefaciens suspension was added
and incubated with the leaf discs for 10 minutes. Leaf discs were
transferred to co-cultivation plates (1/2MS medium) and discs were
placed upside down in contact with filter paper overlaid on the
co-cultivation TOM medium (MS medium with 20 g sucrose/L; 1 mg/L
IAA and 2.5 mg/L BAP). The plate was sealed with parafilm and
labeled appropriately.
Plates were incubated in dim light (60-80 mE/ms) under 18/6
photoperiods at 24.degree. C. for three days. Leaf discs were
transferred to regeneration/selection medium plates with TOM K
media (TOM medium with 300 mg/L kanamycin) and sub-cultured
bi-weekly in the same fresh medium in dim light at 24.degree. C.
until shoots become excisable. Shoots from leaves were removed with
forceps and inserted in MS basal medium with 100 mg/L kanamycin for
rooting at 24.degree. C. under 18/6 photoperiods in dim light
(60-80 mE/ms). When plantlets having both shoots and roots grew
large enough (e.g., reached over half of the height of the GA7
box), they were transferred to soil for acclimatization. During the
transfer, any gel remaining on the root tissue was washed off with
tap water. Established seedlings were transferred to the greenhouse
to set seed and for further analysis.
An RNAi sequence against TLA2 is provided in SEQ ID NO:27. The
sense, spacer and antisense portions of the RNAi molecule are
provided in SEQ ID NOs: 45-47, respectively. An RNAi sequence
against TLA3 is provided in SEQ ID NO:28. The sense, spacer and
antisense portions of the RNAi molecule are provided in SEQ ID NOs:
48-50, respectively. An RNAi sequence against TLA4 is provided in
SEQ ID NO:29. The sense, spacer and antisense portions of the RNAi
molecule are provided in SEQ ID NOs: 51-53, respectively. An RNAi
sequence against CAO-2, CAO-3 and CAO-4 is provided in SEQ ID
NO:30. The sense, spacer and antisense portions of the RNAi
molecule are provided in SEQ ID NOs: 54-56, respectively.
FIGS. 1, 2 and 3 are photographs of the transgenic plants described
herein. FIG. 1A shows a T0 transgenic tobacco plant containing a
TLA2 RNAi construct (right) next to a wild type tobacco plant
(left), while FIG. 1B shows a T1 transgenic tobacco plant
containing a TLA2 RNAi construct. FIG. 2A shows T0 transgenic
tobacco plants containing different integration events for a TLA3
RNAi construct (the three plants on the left) next to a wild type
tobacco plant (the plant on the right). FIGS. 2B and 2C show two T1
transgenic tobacco plants containing a TLA3 RNAi construct. FIG. 3
shows transgenic tobacco plants containing a TLA4 RNAi construct
(right) next to wild type tobacco plants (left).
FIGS. 1, 2 and 3 shows that transgenic tobacco plants expressing a
RNAi construct against a TLA gene exhibit lower chlorophyll levels
depends on the target gene and integration event. As described
herein, chlorophyll levels can be qualitatively determined by
observing the color of the leaf and/or quantitatively determined by
measuring the amount of total chlorophyll and/or the ratio of
chlorophyll a to chlorophyll b.
Example 4--Real Time PCR Confirmation and Western Blot Evaluation
on TLA RNAi Transgenic Lines
RealTime PCR analysis: To confirm the expression pattern of
selected candidate genes, relative changes in transcripts from 16
different samples were measured. In brief, total RNA was isolated
using TM Reagent (Sigma-Aldritch). To remove DNA impurities,
purified RNA was treated with RNase free DNase (Turbo DNA-free;
Ambion). To synthesize the first cDNA strand, approximately 10
.mu.g of total RNA was transcribed utilizing the High Capacity cDNA
Kit (Applied Biosystems). To measure the level of selected gene
transcripts in the samples, RT PCR was performed using SYBR Green
PCR Master Mix (Applied Biosystems). Gene specific primers are
shown below.
Antigenic domains were identified from the sequences of the TLA2,
TLA3 and TLA4 proteins. The oligopeptides shown in the Table below
were synthesized and injected into rabbit to generate polyclonal
antibodies. Western blots then were used to confirm the protein
expression level for the target knock down genes.
Oligopeptides (solid underlining or dashed underlining represents
different charges for the amino acids), and the ratio is the ratio
of polar amino acids to total amino acids:
TABLE-US-00003 TLA 2 ##STR00001## to positions 178-197 of SEQ ID
NO:12] (11:20) ##STR00002## to positions 330-352 of SEQ ID NO:12]
(7:23) TLA 3 ##STR00003## to positions 275-294 of SEQ ID NO:16]
(13:20) ##STR00004## to positions 158-179 of SEQ ID NO:16] (8:22)
TLA 4 ##STR00005## to positions 538-557 of SEQ ID NO: 20] (14:20)
##STR00006## positions 279-296 of SEQ ID NO: 20] (8:18)
TABLE-US-00004 TABLE 3 Real time PCR primers list SEQ ID
Designation Sequence (5' to 3') NO: TLA2-F1
ATGGCTTCTCTATTATCTTCTCGTCTC 38 TLA2-R1 GTTCAAATGCTCAGCTGGTGGAACG 39
TLA3-F1 CGTCAATTCCTCTCTCTCCCGCCTC 40 TLA3-R1
CTTCAGAACCAGCAGCAACAAGCAG 41 TLA4-F1 ATGGAAGCCACTGCTTCTTTCTCCTC 42
TLA4-R1 CTCATTCACTAGGGATACATGGAGGGTG 43
Total tobacco leaf protein extracts from TLA3-RNAi transgenic
plants (e.g., TLA3-1, 691 WT, and TLA3-18) were loaded (on the
basis of equal Ch1) and run on an SDS-PAGE gel. Western blotting
was performed on the SDS-PAGE gel and the membrane was probed with
specific polyclonal antibodies raised against two TLA3
oligopeptides (primary anti-TLA3 antibody diluted at 1:500).
Substantially lower amounts of the TLA3 protein was observed in the
extracts from the TLA3-1 and TLA3-18 plants compared to that of the
wild type plants.
Interestingly, the TLA3 protein from N. tabacum has a predicted
molecular weight of 35 kD, but, under electrophoretic conditions,
the protein migrates to a position of about 25 kD. This faster
electrophoretic mobility is attributed to the fact that TLA3 from
N. tabacum has about 45 negatively charged amino acids and, thus,
migrates faster under the influence of the electrophoresis
field.
FIG. 4A is a graph showing the real time PCR results from T0
generation plants transgenic for TLA2 RNAi. The T2-1, T2-2, T2-3,
T2-4 and T2-6 designations represent individual TLA2 RNAi
transgenic plants while CK refers to a transgenic control tobacco
plant (transformed with an empty vector). The Y axis (2{circumflex
over ( )}-.DELTA.Ct) shows the relative expression level, based on
mRNA levels, of the TLA2 gene. There were different levels of
knock-down of the TLA2 gene depending upon the particular
integration event. Among them, the T2-6 line exhibited plants with
stunted growth; most of the plants died before they matured.
Overall, regenerating transgenic tobacco plants deficient in TAL2
was difficult, and the growth was inhibited by the level of TLA2
knock-down.
FIG. 4B is a graph showing the real time PCR results in T0
generation plants transgenic for TLA3 RNAi. T3-1 to T3-10
designations represent individual TLA3 RNAi transgenic plants,
whereas CK refers to transgenic control tobacco (e.g., transformed
with an empty vector). As with TLA2 transformants, there were
different levels of knock down of TLA3 gene in TLA3 RNAi
transformants.
Example 5--Phenotype Evaluation on TLA RNAi Transgenic Lines
According to the TLA concept proposed by the University of
California--Berkeley group (Polle et al., 2003, Planta, 217:49-59;
Kirst et al., 2012, Plant Physiol., 158:930-45; and Kirst et al.,
2012, Plant Physiol., 160:2251-60), the mutants described herein
should result in smaller light-harvesting chlorophyll antenna size
and a substantially improved photosynthetic efficiency, as well as
a higher [chlorophyll a/chlorophyll b] ratio. The chlorophyll from
tobacco leaf samples was extracted in 80% acetone, and cell debris
was removed by centrifugation at 20,000.times.g for 5 min. The
absorbance of the supernatant was measured with a Shimadzu UV-1800
spectrophotometer, and the Chl concentration of the samples was
determined according to Arnon (1949, Plant Physiol., 24:1-15), with
equations corrected as described by Melis et al. (1989, Philos.
Trans. R. Soc. Lond. B, 323:397-409). Total carotenoid content was
determined according to the method of Lichtenthaler (1987, Methods
Enzymol., 148:350-82).
The antenna size of tobacco leaf samples was calculated by
measuring photosynthetic activity. The oxygen evolution activity of
the tobacco leaf (punched from fresh leaf tissue sample) was
measured at 25.degree. C. with a Clark-type oxygen electrode
illuminated with light from a halogen lamp projector. A Corning
3-69 filter (510-nm cutoff filter) defined the yellow actinic
excitation via which photosynthesis measurements were made. Samples
of 5-mL cell suspension containing 1.3 mM Chl were loaded into the
oxygen electrode chamber. Sodium bicarbonate (100 mL of 0.5 M
solution, pH 7.4) was added to the cell suspension prior to the
oxygen evolution measurements to ensure that oxygen evolution was
not limited by the carbon supply available to the cells. After
registration of the rate of dark respiration by the cells, samples
were illuminated with gradually increasing light intensities. The
rate of oxygen exchange (uptake or evolution) under each of these
irradiance conditions was recorded continuously for a period of
about 5 min.
The following Table shows total chlorophyll measurement and
Chlorophyll a/Chlorophyll b ratios of T0 lines transgenic for TLA2,
TLA3 or TLA4 RNAi.
TABLE-US-00005 TABLE 4 Chlorophyll measurements Chl a Chl b total
Chl Chl a/b Sample (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) Ratio BW1*
17.96 4.29 22.240 4.190 BM2* 5.56 0.61 6.165 9.190 Wild type 43.07
11.40 54.465 3.780 TLA2-1 29.90 5.97 35.860 5.012 TLA2-2 26.40 6.56
32.950 4.027 TLA2-3 5.93 1.29 7.215 4.615 TLA2-4 20.34 4.68 25.015
4.351 TLA2-5 50.73 13.85 64.570 3.664 TLA2-6 18.78 4.76 23.535
3.944 TLA2-7 24.09 5.55 29.640 4.341 TLA3-1 29.53 6.66 36.185 4.433
TLA3-2 45.05 11.00 56.040 4.097 TLA3-3 43.16 6.10 49.255 7.081
TLA3-4 36.73 9.04 45.770 4.063 TLA3-5 38.05 7.22 45.265 5.274
TLA3-6 27.72 5.01 32.720 5.537 TLA3-7 30.27 5.27 35.540 5.744
TLA3-8 53.82 13.29 67.105 4.049 TLA3-9 29.61 4.95 34.555 5.988
TLA3-10 24.09 4.45 28.535 5.420 TLA3-11 36.04 5.31 41.340 6.793
TLA3-12 28.50 4.27 32.770 6.674 TLA3-13 33.61 5.63 39.235 5.969
TLA3-14 22.65 3.75 26.400 6.040 TLA3-15 21.62 3.29 24.910 6.571
TLA3-16 27.80 5.77 33.565 4.817 TLA3-17 33.03 6.90 39.920 4.790
TLA3-18 27.34 7.35 34.690 3.720 TLA3-19 30.06 6.67 36.725 4.510
TLA3-20 23.56 4.44 27.995 5.305 TLA3-21 21.25 2.89 24.135 7.366
TLA3-22 22.28 4.17 26.450 5.343 TLA4-1 8.90 2.07 10.960 4.308
TLA4-2 46.74 12.06 58.790 3.877 TLA4-3 29.90 5.97 35.860 5.012
TLA4-4 21.04 4.83 25.870 4.356 TLA4-5 39.16 8.72 47.875 4.493
TLA4-6 38.63 7.88 46.505 4.902 *BW1: UC Berkley wild type line;
BM2: UC Berkley TLA mutant line
Tobacco lines exhibit a particular ratio of chlorophyll a/b, which
can be used as an index for total antenna size. Therefore, changes
in the chlorophyll a/b ration can be used to measure changes in
antenna size. As shown in the Table above, the UC Berkeley mutant
line increased the chlorophyll a/b ratio to about 9, from a wild
type ratio of about 4. In the NLM tobacco lines described herein,
the wild type ratio of chlorophyll a/b is about 4, but in T0
generations of NLM plants transgenic for a TLA3 RNAi, most of the
mutant lines reached a chlorophyll a/b ratio of about 5 to about 8.
There was no obvious increase in chlorophyll a/b ratio in the T0
generation of plants transgenic for TLA2 RNAi and TLA4 RNAi. These
results indicate that TLA3 likely is the initial candidate gene to
knock down and decrease antenna size in photosynthetic light
harvesting centers in tobacco.
T1 generation of TLA3 RNAi line 1 (501-1 to 501-24) and 3 (data not
shown) were harvested and chlorophyll a/b ratios were determined.
The following Table shows chlorophyll data for the T1 generation of
the TLA3 RNAi plants. The chlorophyll a/b ratios in the T1
generation of the TLA3 RNAi plants were higher compared to wild
type ratios, and the change in the ratio was stable in the T1
generation.
TABLE-US-00006 TABLE 5 Chlorophyll measurements Chl a Chl b total
Chl Chl a/b Sample (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) Ratio 503_1
19.48 2.58 22.06 7.57 503_2 31.14 6.85 37.99 4.55 503_3 32.94 7.37
40.32 4.47 503_4 31.71 6.59 38.30 4.81 503_5 44.48 8.26 52.74 5.38
503_6 44.88 9.67 54.56 4.64 503_7 49.16 10.74 59.89 4.58 503_8
38.81 8.09 46.90 4.80 503_9 40.97 9.89 50.86 4.14 503_10 33.30 7.27
40.57 4.58 503_11 29.03 5.86 34.89 4.95 503_12 33.10 7.77 40.87
4.26 503_13 30.27 5.62 35.88 5.39 503_14 34.49 6.89 41.38 5.01
503_15 36.55 7.74 44.28 4.72 503_16 34.80 7.00 41.79 4.97 503_17
42.21 8.61 50.81 4.90 503_18 20.70 3.24 23.93 6.39 503_19 38.04
8.50 46.54 4.48 503_20 38.30 7.79 46.08 4.92 503_21 29.45 4.86
34.31 6.06 503_22 34.28 8.08 42.36 4.24 503_23 29.14 5.79 34.92
5.04 503_24 28.36 8.96 37.31 3.17 Wild type 46.61 13.67 60.27
3.41
Antenna size of the photosynthetic light harvesting centers (both
PSI and PSII) were measured in the T1 generation for TLA3 RNAi
plants, TLA2 RNAi plants, and TLA4 RNAi plants. The following Table
shows the antenna size measurement of PSI and PSII in the T1
generation for four TLA2 RNAi line 2 plants (e.g., 2-1-1, 2-1-2,
2-1-3, and 2-1-4). The data showed that total antenna size in PSI
for mutant lines was similar to wild type, but that PSII antenna
size decreased in TLA2 knock out lines.
TABLE-US-00007 TABLE 6 Chlorophyll and antenna measurements PSII
PSI Chl a Chl b Total Total Chl a/b Chl/Car antenna size antenna
size Sample content content Chl Car ratio ratio (molecules
(molecule number (.mu.g/ml) (.mu.g/ml) (.mu.g/ml) (.mu.g/ml)
(mol:mol) (mol:mol) in PSII) in PSI) WT 1 18.84 5.93 24.77 5.48
3.18 4.52 215 180 (2.70*) (5.54*) WT 2 25.37 7.94 33.30 6.64 3.20
5.02 185 197 (2.73*) (5.98*) 2-1-1 18.13 5.29 23.42 4.93 3.43 4.75
164 178 (3.02*) (6.16*) 2-1-2 11.59 3.81 15.40 2.57 3.04 6.00 125
168 (2.88*) (6.56*) 2-1-3 17.90 4.73 22.63 4.48 3.79 5.05 125 184
(3.02*) (6.66*) 2-1-4 12.23 4.26 16.48 2.76 2.87 5.98 125 166
(2.92*) (6.24*) *ratios measured in isolated thylakoids
The antenna size for PSI and PSII in the T1 generation of TLA3 RNAi
line 3 plants (e.g., 3-3-1, 3-3-2, 3-3-3, 3-3-4, 3-3-5, 3-3-6, and
3-3-7) is shown in the following Table. Notably, the total number
of photoreceptor antenna was knocked down in the transgenic plants
from 215 to 160 for PSII and from 195 to 160 for PSI. The decrease
in antenna size was correlated with the increase in chlorophyll a/b
ratio in the T1 generation.
TABLE-US-00008 TABLE 7 Chlorophyll and antenna measurements PSII
PSI antenna size antenna size Chl a Chl b Tot Chl* Total Car Chl
a/b Chl/Car (molecules (molecules Sample [.mu.g/ml] [.mu.g/ml]
[.mu.g/ml] [.mu.g/ml] (mol:mol) (mol:mol) in PSII) in PSII) WT 1
32.1 11 43.1 7.91 2.91 5.46 215 195 WT 2 26.6 8.83 35.5 6.29 3.02
5.64 3-3-1 16.8 3.68 20.5 4.4 4.56 4.65 160 164 3-3-2 15.1 3.43
18.5 4.12 4.4 4.49 3-3-3 13.9 3.26 17.1 3.4 4.26 5.04 3-3-4 14.6
3.36 18 3.57 4.34 5.03 3-3-5 15.8 3.54 19.4 4.66 4.47 4.16 148 162
3-3-6 32.1 11 43.1 7.91 2.91 5.46 215 195 3-3-7 26.6 8.83 35.5 6.29
3.02 5.64
The following Table shows antenna size in PSI and PSII from T1
generation plants transgenic for TLA4 RNAi line 2 and line 6. This
data demonstrated that total antenna size of the mutant lines was
similar to that of the wild type in both PSI and PSII.
TABLE-US-00009 TABLE 8 Chlorophyll and antenna measurements PSII
PSI Sample Chl a Chl b Tot Chl* Total Car Chl a/b Chl/Car antenna
size antenna size WT 1 24.1 8.0 32.0 5.40 3.00 5.86 210 WT 2 27.3
9.2 36.5 6.29 2.93 6.03 200 195 TLA4-2-1 30.1 10.8 41.0 6.30 2.76
6.48 TLA4-2-2 32.9 12.0 45.0 6.80 2.72 6.55 232 TLA4-2-4 29.3 11.1
40.5 6.10 2.63 6.59 190 TLA4-6-1 29.3 11.1 40.5 6.10 2.63 6.59 156
165 TLA4-6-2 27.9 9.7 37.7 6.30 2.85 5.90 230 TLA4-6-3 25.7 10.2
36.0 5.40 2.52 6.64 185 145
Example 6--CAO RNAi Plant Generation
Tobacco plants transgenic for the CAO RNAi nucleic acid shown in
SEQ ID NO:30 were produced as described herein, and chlorophyll a
and chlorophyll b was measured as described herein. The data is
shown in the following Table.
TABLE-US-00010 TABLE 9 Chlorophyll measurements Chl A Chl B Total
Chl a/b Sample (.mu.g/mL) (.mu.g/mL) (.mu.g/mL) Ratio Wild type
17.56 4.42 21.97 3.97 CAOi-1 15.75 2.49 18.23 6.33 CAOi-2 12.73
1.78 14.50 7.16
This data demonstrated that antenna size of the mutant lines was
decreased relative to that of the wild type plants. FIG. 5A shows a
T0 generation tobacco plant transgenic for CAOi-1, and FIG. 5B
shows a T0 generation tobacco plant transgenic for CAOi-2.
Example 7--Phenotypes of TLA3 RNAi Plants
Decreasing, or truncating, the chlorophyll antenna size of the
photosystems should improve photosynthetic solar energy conversion
efficiency and productivity in mass cultures of algae or plants by
up to 3-fold. A Truncated Light-harvesting chlorophyll Antenna size
(TLA) in photosynthetic organisms should help alleviate excess
absorption of sunlight and the ensuing wasteful non-photochemical
dissipation of excitation energy and, thus, would increase
solar-to-biomass energy conversion efficiency and photosynthetic
productivity in high density cultures.
Tobacco was grown under conditions that result in high-density
canopies to evaluate the TLA plants described herein. The T0 stage
of multiple NL Madole TLA3-RNAi transformants were screened and
selected to identify lines for generating T1 seeds. The latter were
germinated and T1 leaves were subjected to phenotypic and
functional analysis. Plants were grown in high density canopies,
with the canopy layout of 25 plants in a 5.times.5 configuration,
and the distance between individual plants set at 9 inches.
Biochemical analysis and biomass accumulation was performed.
This work showed a 25% improvement in stem and leaf biomass
accumulation for the TLA tobacco canopies over that of their
wild-type counterparts grown under the same ambient conditions.
Distinct differences were observed in the appearance of the canopy
between plants containing a TLA RNAi and wild type tobacco plants.
For example, the TLA3-RNAi canopy was a light-green color, while
the wild type canopy was a much darker green. The results described
herein can lead to significant improvements in agronomy,
agricultural productivity, and the optimization of photosynthesis
in commodity crops (e.g., tobacco) or parts thereof (e.g.,
leaves).
The average biomass values were determined for four different
canopies. The results demonstrated that canopy interior plants
performed better than plants in the periphery, as would be expected
from the greater transmittance of sunlight. Significantly, an
increase in leaf biomass of 10.2% was observed for the canopy
interior TLA3-RNAi plants as compared to that of the corresponding
wild type plants.
It is to be understood that, while the methods and compositions of
matter have been described herein in conjunction with a number of
different aspects, the foregoing description of the various aspects
is intended to illustrate and not limit the scope of the methods
and compositions of matter. Other aspects, advantages, and
modifications are within the scope of the following claims.
Disclosed are methods and compositions that can be used for, can be
used in conjunction with, can be used in preparation for, or are
products of the disclosed methods and compositions. These and other
materials are disclosed herein, and it is understood that
combinations, subsets, interactions, groups, etc. of these methods
and compositions are disclosed. That is, while specific reference
to each various individual and collective combinations and
permutations of these compositions and methods may not be
explicitly disclosed, each is specifically contemplated and
described herein. For example, if a particular composition of
matter or a particular method is disclosed and discussed and a
number of compositions or methods are discussed, each and every
combination and permutation of the compositions and the methods are
specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed.
SEQUENCE LISTINGS
1
56125DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1atggcttctc tattatcttc tcgtc 25224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2tcatgggaag atagcattaa caaa 24328DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 3atggcttctc tattatcttc
tcgtctcc 28425DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 4ttaaatagaa tctgccacat aaatg
25526DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5atggatgctc tcttcgtcaa ttcctc 26623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gcccttgttc aacttactac att 23725DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 7atggatgctc tgttcgtcaa
ttcct 25825DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8tcaacttact acattctgtt gaccc 25927DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9atggaagcca ctgcttcttt ctcctca 271026DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10ttagttcttg gccccaaaac cacgag 26111116DNANicotiana tabacum
11atggcttctc tattatcttc tcgtctccca cgtcatcttt cctctaataa accggtactc
60ccaccatcaa gctccggttc aaatctcctt cacaacttca catataaaac ccggttcgat
120caatcccggt tcaaatgctc agctggtgga acggggttct tcacgaagtt
gggtcgtttg 180ctgaaagaga aagcaaagag cgacgtggag aaactgttct
caggattctc aaaaactcga 240gacaatttag cagttataga tgaactcctc
ctttactgga acctttctga cactgaccgt 300gttcttgatg aacttgaaga
ggttctgttg gtgtctgatt ttggcccgaa gattaccata 360aagattgtgg
agagcttgcg ggaggatata tatgggggga aaatcaaatc aggaagtgag
420attaaaagtg ctcttaagaa gagtatcttg gatctattga ctagcaaggc
acctaaaaca 480gagctccgtc tgggcttcag gaaaccatct gtgatcatga
ttgtgggcgt caacggaggt 540gggaagacaa catctcttgg aaagctggca
aatagattga agaaagaagg ggctaagata 600ctattagcag ctggtgatac
atttagagca gctgctagtg atcagttaga aatttgggct 660gaaaggactg
gttgtgagat cgttgttgct gaaaaagaga aagctaaggc atcatcagtt
720ctttcgcagg ctgttaaaag aggaaaggaa gagggtttcg atattgtttt
atgcgacaca 780tctggccgtc tgcacaccta ctatagcttg atggaggaat
tggtggcatg caaaaaagtt 840gtcagtaaaa ttgttactgg tgcacctaat
gaaatcttgc ttgtactgga tggaactact 900ggtttaaata tgcttccaca
agcaagagag tttaacgatg ttgttggagt cactggctta 960atattgacta
aacttgatgg ttctgctcga ggtggctgtg tggttagtgt ggttgatgaa
1020cttggcattc ctgtaaagtt tgtaggtgtt ggggaaggtg tagatgacct
ccaaccgttc 1080aatgctgagg aatttgttaa tgctatcttc ccatga
111612371PRTNicotiana tabacum 12Met Ala Ser Leu Leu Ser Ser Arg Leu
Pro Arg His Leu Ser Ser Asn1 5 10 15Lys Pro Val Leu Pro Pro Ser Ser
Ser Gly Ser Asn Leu Leu His Asn 20 25 30Phe Thr Tyr Lys Thr Arg Phe
Asp Gln Ser Arg Phe Lys Cys Ser Ala 35 40 45Gly Gly Thr Gly Phe Phe
Thr Lys Leu Gly Arg Leu Leu Lys Glu Lys 50 55 60Ala Lys Ser Asp Val
Glu Lys Leu Phe Ser Gly Phe Ser Lys Thr Arg65 70 75 80Asp Asn Leu
Ala Val Ile Asp Glu Leu Leu Leu Tyr Trp Asn Leu Ser 85 90 95Asp Thr
Asp Arg Val Leu Asp Glu Leu Glu Glu Val Leu Leu Val Ser 100 105
110Asp Phe Gly Pro Lys Ile Thr Ile Lys Ile Val Glu Ser Leu Arg Glu
115 120 125Asp Ile Tyr Gly Gly Lys Ile Lys Ser Gly Ser Glu Ile Lys
Ser Ala 130 135 140Leu Lys Lys Ser Ile Leu Asp Leu Leu Thr Ser Lys
Ala Pro Lys Thr145 150 155 160Glu Leu Arg Leu Gly Phe Arg Lys Pro
Ser Val Ile Met Ile Val Gly 165 170 175Val Asn Gly Gly Gly Lys Thr
Thr Ser Leu Gly Lys Leu Ala Asn Arg 180 185 190Leu Lys Lys Glu Gly
Ala Lys Ile Leu Leu Ala Ala Gly Asp Thr Phe 195 200 205Arg Ala Ala
Ala Ser Asp Gln Leu Glu Ile Trp Ala Glu Arg Thr Gly 210 215 220Cys
Glu Ile Val Val Ala Glu Lys Glu Lys Ala Lys Ala Ser Ser Val225 230
235 240Leu Ser Gln Ala Val Lys Arg Gly Lys Glu Glu Gly Phe Asp Ile
Val 245 250 255Leu Cys Asp Thr Ser Gly Arg Leu His Thr Tyr Tyr Ser
Leu Met Glu 260 265 270Glu Leu Val Ala Cys Lys Lys Val Val Ser Lys
Ile Val Thr Gly Ala 275 280 285Pro Asn Glu Ile Leu Leu Val Leu Asp
Gly Thr Thr Gly Leu Asn Met 290 295 300Leu Pro Gln Ala Arg Glu Phe
Asn Asp Val Val Gly Val Thr Gly Leu305 310 315 320Ile Leu Thr Lys
Leu Asp Gly Ser Ala Arg Gly Gly Cys Val Val Ser 325 330 335Val Val
Asp Glu Leu Gly Ile Pro Val Lys Phe Val Gly Val Gly Glu 340 345
350Gly Val Asp Asp Leu Gln Pro Phe Asn Ala Glu Glu Phe Val Asn Ala
355 360 365Ile Phe Pro 37013966DNANicotiana tabacum 13atggcttctc
tattatcttc tcgtctccca catcatcttt cctctaataa accggtactc 60ccaccatcaa
gctccggttc aaatctcctt cacaacttca catataaaac ccggttcgat
120caatcccggt tcaaatgctc agctggtgga acggggttct tcacgaagtt
gggtcgtttg 180ctgaaagaga aagcaaagag cgacgtggag aaactgttct
caggattctc aaaaactcga 240gacaatttag cagttataga tgaactcctc
ctttactgga acctttctga cactgaccgt 300gttcttgatg aacttgaaga
ggttctgttg gtgtctgatt ttggcccgaa gattaccata 360aagattgtgg
agagcttgcg ggaggatata tatgggggga aaatcaaatc aggaagtgag
420attaaaagtg ctcttaagaa gagtatcttg gatctattga ctagcaaggc
acctaaaaca 480gagctccgtc tgggcttcag gaaaccatct gtgatcatga
ttgtgggcgt caacggaggt 540gggaagacaa catctcttgg aaagctggca
aatagattga agaaagaagg ggctaagata 600ctattagcag ctggtgatac
atttagagca gctgctagtg atcagttaga aatttgggct 660gaaaggactg
gttgtgagat cgttgttgct gaaaaagaga aagctaaggc atcatcagtt
720ctttcgcagg ctgttaaaag aggaaaggaa gagggtttcg atattgtttt
atgcgacaca 780tctggccgtc tgcacaccta ctatagcttg atggaggaat
tggtggcatg caaaaaagtt 840gtcagtaaaa ttgttactgg tgcacctaat
aggagcagta tacaacagga actaagatta 900gcagcttgct actactctga
aaattcaaca ttcacaaata acatttatgt ggcagattct 960atttaa
96614320PRTNicotiana tabacum 14Met Ala Ser Leu Leu Ser Ser Arg Leu
Pro His His Leu Ser Ser Asn1 5 10 15Lys Pro Val Leu Pro Pro Ser Ser
Ser Gly Ser Asn Leu Leu His Asn 20 25 30Phe Thr Tyr Lys Thr Arg Phe
Asp Gln Ser Arg Phe Lys Cys Ser Ala 35 40 45Gly Gly Thr Gly Phe Phe
Thr Lys Leu Gly Arg Leu Leu Lys Glu Lys 50 55 60Ala Lys Ser Asp Val
Glu Lys Leu Phe Ser Gly Phe Ser Lys Thr Arg65 70 75 80Asp Asn Leu
Ala Val Ile Asp Glu Leu Leu Leu Tyr Trp Asn Leu Ser 85 90 95Asp Thr
Asp Arg Val Leu Asp Glu Leu Glu Glu Val Leu Leu Val Ser 100 105
110Asp Phe Gly Pro Lys Ile Thr Ile Lys Ile Val Glu Ser Leu Arg Glu
115 120 125Asp Ile Tyr Gly Gly Lys Ile Lys Ser Gly Ser Glu Ile Lys
Ser Ala 130 135 140Leu Lys Lys Ser Ile Leu Asp Leu Leu Thr Ser Lys
Ala Pro Lys Thr145 150 155 160Glu Leu Arg Leu Gly Phe Arg Lys Pro
Ser Val Ile Met Ile Val Gly 165 170 175Val Asn Gly Gly Gly Lys Thr
Thr Ser Leu Gly Lys Leu Ala Asn Arg 180 185 190Leu Lys Lys Glu Gly
Ala Lys Ile Leu Leu Ala Ala Gly Asp Thr Phe 195 200 205Arg Ala Ala
Ala Ser Asp Gln Leu Glu Ile Trp Ala Glu Arg Thr Gly 210 215 220Cys
Glu Ile Val Val Ala Glu Lys Glu Lys Ala Lys Ala Ser Ser Val225 230
235 240Leu Ser Gln Ala Val Lys Arg Gly Lys Glu Glu Gly Phe Asp Ile
Val 245 250 255Leu Cys Asp Thr Ser Gly Arg Leu His Thr Tyr Tyr Ser
Leu Met Glu 260 265 270Glu Leu Val Ala Cys Lys Lys Val Val Ser Lys
Ile Val Thr Gly Ala 275 280 285Pro Asn Arg Ser Ser Ile Gln Gln Glu
Leu Arg Leu Ala Ala Cys Tyr 290 295 300Tyr Ser Glu Asn Ser Thr Phe
Thr Asn Asn Ile Tyr Val Ala Asp Ser305 310 315
320151121DNANicotiana tabacum 15atggatgctc tcttcgtcaa ttcctctctc
tcccgcctca aactcaaatt ctcccctcaa 60ttccctccca ccttctctca tcaacctttt
atctgtctaa agaaactcgg caataagaac 120aatttatcag tatttgctac
gcttcagaac cagcagcaac aagcagtcga agcagcagaa 180gacgaagaac
cggtactgtt tgaagattac gatgaggatg aaacgtacgg agaagttaac
240aaaattatcg gcagtcgagc aattgaacgt gggaaaggaa tggagtactt
gatagagtgg 300aaagacgaac atgccccaac gtgggtcccc tctgattaca
ttgctaaaga tgttgtggcc 360gagtacgaaa ctccttggtg gaatgcggct
aaaaaggccg acgaatccgc tcttagggaa 420ctcctagaaa ctgacgacga
cagagatgtg gacgcagtag atgaggatgg acgtacggct 480ttgctctttg
tctcgggtct ggggtccgag ccgtgtgtca agctgctagc tgaagccggc
540gctgacgtgg actatcgcga taggaatggc ggcttgactg ctctgcatat
ggcagccggc 600tatgttaagc cgggtgtcgc caagctgtta attgacctcg
gggcagaccc cgaggtcgag 660gattatagag gacaaacgcc tctgagcttg
gcgaggatga ttttgaatca aacgcctaaa 720ggaaatccaa tgcaattcgc
gaggagattg gggttagaga atgtggttag gattttggag 780gatgcgattt
tcgagtatgc aacagtggag gaaatattgg agaagagagg gaaaggcgaa
840aatgtggagt atttagtgaa gtggaaggat ggggaggata acgagtgggt
caaagcatgg 900ctgatatctg aagatttggt gagggatttt gaggctggtt
tggaatatgc agtagcagat 960tgtattcttg agaagagaga aggtgaggat
gggaagggag aatacttggt taaatggact 1020gatattgagg aagctacgtg
ggaacccgaa gaaaatgttg acccccttct tatagaagat 1080tttgaaaaga
gtcaacagaa tgtagtaagt tgaacaaggg c 112116370PRTNicotiana tabacum
16Met Asp Ala Leu Phe Val Asn Ser Ser Leu Ser Arg Leu Lys Leu Lys1
5 10 15Phe Ser Pro Gln Phe Pro Pro Thr Phe Ser His Gln Pro Phe Ile
Cys 20 25 30Leu Lys Lys Leu Gly Asn Lys Asn Asn Leu Ser Val Phe Ala
Thr Leu 35 40 45Gln Asn Gln Gln Gln Gln Ala Val Glu Ala Ala Glu Asp
Glu Glu Pro 50 55 60Val Leu Phe Glu Asp Tyr Asp Glu Asp Glu Thr Tyr
Gly Glu Val Asn65 70 75 80Lys Ile Ile Gly Ser Arg Ala Ile Glu Arg
Gly Lys Gly Met Glu Tyr 85 90 95Leu Ile Glu Trp Lys Asp Glu His Ala
Pro Thr Trp Val Pro Ser Asp 100 105 110Tyr Ile Ala Lys Asp Val Val
Ala Glu Tyr Glu Thr Pro Trp Trp Asn 115 120 125Ala Ala Lys Lys Ala
Asp Glu Ser Ala Leu Arg Glu Leu Leu Glu Thr 130 135 140Asp Asp Asp
Arg Asp Val Asp Ala Val Asp Glu Asp Gly Arg Thr Ala145 150 155
160Leu Leu Phe Val Ser Gly Leu Gly Ser Glu Pro Cys Val Lys Leu Leu
165 170 175Ala Glu Ala Gly Ala Asp Val Asp Tyr Arg Asp Arg Asn Gly
Gly Leu 180 185 190Thr Ala Leu His Met Ala Ala Gly Tyr Val Lys Pro
Gly Val Ala Lys 195 200 205Leu Leu Ile Asp Leu Gly Ala Asp Pro Glu
Val Glu Asp Tyr Arg Gly 210 215 220Gln Thr Pro Leu Ser Leu Ala Arg
Met Ile Leu Asn Gln Thr Pro Lys225 230 235 240Gly Asn Pro Met Gln
Phe Ala Arg Arg Leu Gly Leu Glu Asn Val Val 245 250 255Arg Ile Leu
Glu Asp Ala Ile Phe Glu Tyr Ala Thr Val Glu Glu Ile 260 265 270Leu
Glu Lys Arg Gly Lys Gly Glu Asn Val Glu Tyr Leu Val Lys Trp 275 280
285Lys Asp Gly Glu Asp Asn Glu Trp Val Lys Ala Trp Leu Ile Ser Glu
290 295 300Asp Leu Val Arg Asp Phe Glu Ala Gly Leu Glu Tyr Ala Val
Ala Asp305 310 315 320Cys Ile Leu Glu Lys Arg Glu Gly Glu Asp Gly
Lys Gly Glu Tyr Leu 325 330 335Val Lys Trp Thr Asp Ile Glu Glu Ala
Thr Trp Glu Pro Glu Glu Asn 340 345 350Val Asp Pro Leu Leu Ile Glu
Asp Phe Glu Lys Ser Gln Gln Asn Val 355 360 365Val Ser
370171113DNANicotiana tabacum 17atggatgctc tgttcgtcaa ttcctctctc
tcccgcctca aactcaaatt ctcccctcaa 60ttccctccca ccttctctca tcaacctttt
atccgtctaa agaaactcgg caacaagaac 120aatttctcag tatttgctac
gcttcagaac cagcagcaac aagcagtcgc agctgctgaa 180gaggaagaac
cggtactgtt tgaagattac gatgaggatg aaacgtacgg agaagttaac
240aaaatcatcg gaagtagagc aattgaaggt gggaaaggaa tggagtactt
gatagagtgg 300aaagacgaac atgccccaac atgggtcccc tctgattaca
ttgctaaaga tgttgtggcc 360gagtacgaaa ctccttggtg gaatgccgct
aaaaaggccg acgaatccgc tcttaagaaa 420tttctagaag ctgacgacga
cagagatgtg gacgcagttg atgaggatgg acgtacggct 480ttgctctttg
tctcgggtct ggggtccgag ccgtgtgtca agctgctagc tgaagctggc
540gctgacgtgg actatcgcga taggaatggc ggcttgacgg ctctgcacat
ggcagccggc 600tatgttaagc cgggtgtcgc caagctgtta attgacctcg
gggcagaccc tgaggtcgag 660gattatagag gacaaacgcc tctgagcttg
gcgaggatga ttttgaatca aacgcctaaa 720ggaaacccaa tgcaatttgc
taggagattg ggactagaga atgtggttag gatattggag 780gatgcgattt
tcgaatatgc aacagtggag gagatattgg agaagagagg gaaaggtgaa
840aatgtggagt atttagtcaa gtggaaggat ggggaggata atgaatgggt
gaaagcatgg 900ctgataagtg aggatttggt gagagatttt gaggctggtt
tggaatatgc agtggcagag 960tgtattcttg agaagagaga aggtgaggat
gggaagggag aatatttggt taaatggact 1020gatattgagg aagctacctg
ggaaccggaa gaaaatgttg acccccttct aatagaagat 1080tttgaaaagg
gtcaacagaa tgtagtaagt tga 111318370PRTNicotiana tabacum 18Met Asp
Ala Leu Phe Val Asn Ser Ser Leu Ser Arg Leu Lys Leu Lys1 5 10 15Phe
Ser Pro Gln Phe Pro Pro Thr Phe Ser His Gln Pro Phe Ile Arg 20 25
30Leu Lys Lys Leu Gly Asn Lys Asn Asn Phe Ser Val Phe Ala Thr Leu
35 40 45Gln Asn Gln Gln Gln Gln Ala Val Ala Ala Ala Glu Glu Glu Glu
Pro 50 55 60Val Leu Phe Glu Asp Tyr Asp Glu Asp Glu Thr Tyr Gly Glu
Val Asn65 70 75 80Lys Ile Ile Gly Ser Arg Ala Ile Glu Gly Gly Lys
Gly Met Glu Tyr 85 90 95Leu Ile Glu Trp Lys Asp Glu His Ala Pro Thr
Trp Val Pro Ser Asp 100 105 110Tyr Ile Ala Lys Asp Val Val Ala Glu
Tyr Glu Thr Pro Trp Trp Asn 115 120 125Ala Ala Lys Lys Ala Asp Glu
Ser Ala Leu Lys Lys Phe Leu Glu Ala 130 135 140Asp Asp Asp Arg Asp
Val Asp Ala Val Asp Glu Asp Gly Arg Thr Ala145 150 155 160Leu Leu
Phe Val Ser Gly Leu Gly Ser Glu Pro Cys Val Lys Leu Leu 165 170
175Ala Glu Ala Gly Ala Asp Val Asp Tyr Arg Asp Arg Asn Gly Gly Leu
180 185 190Thr Ala Leu His Met Ala Ala Gly Tyr Val Lys Pro Gly Val
Ala Lys 195 200 205Leu Leu Ile Asp Leu Gly Ala Asp Pro Glu Val Glu
Asp Tyr Arg Gly 210 215 220Gln Thr Pro Leu Ser Leu Ala Arg Met Ile
Leu Asn Gln Thr Pro Lys225 230 235 240Gly Asn Pro Met Gln Phe Ala
Arg Arg Leu Gly Leu Glu Asn Val Val 245 250 255Arg Ile Leu Glu Asp
Ala Ile Phe Glu Tyr Ala Thr Val Glu Glu Ile 260 265 270Leu Glu Lys
Arg Gly Lys Gly Glu Asn Val Glu Tyr Leu Val Lys Trp 275 280 285Lys
Asp Gly Glu Asp Asn Glu Trp Val Lys Ala Trp Leu Ile Ser Glu 290 295
300Asp Leu Val Arg Asp Phe Glu Ala Gly Leu Glu Tyr Ala Val Ala
Glu305 310 315 320Cys Ile Leu Glu Lys Arg Glu Gly Glu Asp Gly Lys
Gly Glu Tyr Leu 325 330 335Val Lys Trp Thr Asp Ile Glu Glu Ala Thr
Trp Glu Pro Glu Glu Asn 340 345 350Val Asp Pro Leu Leu Ile Glu Asp
Phe Glu Lys Gly Gln Gln Asn Val 355 360 365Val Ser
370191692DNANicotiana tabacum 19atggaagcca ctgcttcttt ctcctcaact
atgtcttccc accatttctt tccactttcc 60aaagccaccc tctcaacttc taaacttcca
ttttctggga ctggttcaac tcattctctt 120tcattttctt caagaaactc
attcactagg gatacatgga gggtgatcaa ttcaaggaat 180gtggttattt
caagaagaga aatgcgtgga gttattagag ctgagatgtt tggacagctc
240actagtggac ttgaatcagc ttggaataag ctcaaaggag aagaggtttt
gaccaaggaa 300aacattgtgg aacctatgag agacatcagg
agggctcttt tggaagctga tgttagtctc 360cctgttgtca gaaggtttgt
tcagtctgtt agtgacgaag ccgtggggac tggcttgatt 420cgaggagtaa
gaccagatca gcaactagtt aaaattgtac gagacgagct tgtgaaactg
480atgggtggag aggtctctga actggtattt gctaaatctg gacccaccat
aatactattg 540gccggtctac aaggtgttgg aaagacaact gttagcgcaa
agttagcttt atatctaaag 600aagcagggta agagttgcat gctgattgct
ggagacgtgt atagacctgc tgctattgac 660caacttgtta ttttgggtga
acaggttgat gtgcctgttt atgcagcagg aacagacgta 720aaacctgcag
aaatagcccg tcaaggatta gaagaggcca aaagaaagaa tgtagatgta
780gtcataatgg atacagctgg acgacttcag atagataaag ctatgatgga
tgaattaaaa 840gaggtgaaac gggtactgaa ccccacagag gttttgcttg
ttgtggatgc aatgactggc 900caagaagctg cagctttggt cacaacattc
aatctcgaaa ttggaattac tggtgccatt 960atgacaaagc tagatgggga
ttctaggggt ggagcagctt taagtgtcaa ggaggtatca 1020ggaaagccaa
ttaagctcgt aggaagaggt gaacgtatgg aggaccttga acctttctat
1080cctgaccgca tggctggacg tattttaggg atgggagatg ttctgtcgtt
tgttgaaaaa 1140gcccaagaag ttatgaaaca agaagatgca gaagatttgc
agaagaagat catgagtgca 1200aaatttgatt tcaatgactt cctgaagcaa
actcgtgcag ttgctcaaat gggtaccatg 1260tcccgcgttc tcggaatgat
tcctggcatg ggaaaggtta ctcctgcaca aattcgagag 1320gcagagaaga
gcttaataat aatggagtca atgatagaag tcatgacacc agaggagaag
1380gagaaaccag aactgttagc agaatctcct agtagaagga aacgtattgc
tcaagagtcc 1440gggaaaactg agcagcaggt gagtcaactt gttgctcaac
tttttcaaat gcgtgtacgt 1500atgaagaatt tgatgggtgt tatgcaaggt
ggttccatac ctgcactgag taatcttgag 1560gaggcactta aaactgaaca
gaaggctcct cctggtactg caaggaggaa gcgaaggtca 1620gaatcaagaa
agcaatttgc agactcggga tcaactagac ctggccctcg tggttttggg
1680gccaagaact aa 169220563PRTNicotiana tabacum 20Met Glu Ala Thr
Ala Ser Phe Ser Ser Thr Met Ser Ser His His Phe1 5 10 15Phe Pro Leu
Ser Lys Ala Thr Leu Ser Thr Ser Lys Leu Pro Phe Ser 20 25 30Gly Thr
Gly Ser Thr His Ser Leu Ser Phe Ser Ser Arg Asn Ser Phe 35 40 45Thr
Arg Asp Thr Trp Arg Val Ile Asn Ser Arg Asn Val Val Ile Ser 50 55
60Arg Arg Glu Met Arg Gly Val Ile Arg Ala Glu Met Phe Gly Gln Leu65
70 75 80Thr Ser Gly Leu Glu Ser Ala Trp Asn Lys Leu Lys Gly Glu Glu
Val 85 90 95Leu Thr Lys Glu Asn Ile Val Glu Pro Met Arg Asp Ile Arg
Arg Ala 100 105 110Leu Leu Glu Ala Asp Val Ser Leu Pro Val Val Arg
Arg Phe Val Gln 115 120 125Ser Val Ser Asp Glu Ala Val Gly Thr Gly
Leu Ile Arg Gly Val Arg 130 135 140Pro Asp Gln Gln Leu Val Lys Ile
Val Arg Asp Glu Leu Val Lys Leu145 150 155 160Met Gly Gly Glu Val
Ser Glu Leu Val Phe Ala Lys Ser Gly Pro Thr 165 170 175Ile Ile Leu
Leu Ala Gly Leu Gln Gly Val Gly Lys Thr Thr Val Ser 180 185 190Ala
Lys Leu Ala Leu Tyr Leu Lys Lys Gln Gly Lys Ser Cys Met Leu 195 200
205Ile Ala Gly Asp Val Tyr Arg Pro Ala Ala Ile Asp Gln Leu Val Ile
210 215 220Leu Gly Glu Gln Val Asp Val Pro Val Tyr Ala Ala Gly Thr
Asp Val225 230 235 240Lys Pro Ala Glu Ile Ala Arg Gln Gly Leu Glu
Glu Ala Lys Arg Lys 245 250 255Asn Val Asp Val Val Ile Met Asp Thr
Ala Gly Arg Leu Gln Ile Asp 260 265 270Lys Ala Met Met Asp Glu Leu
Lys Glu Val Lys Arg Val Leu Asn Pro 275 280 285Thr Glu Val Leu Leu
Val Val Asp Ala Met Thr Gly Gln Glu Ala Ala 290 295 300Ala Leu Val
Thr Thr Phe Asn Leu Glu Ile Gly Ile Thr Gly Ala Ile305 310 315
320Met Thr Lys Leu Asp Gly Asp Ser Arg Gly Gly Ala Ala Leu Ser Val
325 330 335Lys Glu Val Ser Gly Lys Pro Ile Lys Leu Val Gly Arg Gly
Glu Arg 340 345 350Met Glu Asp Leu Glu Pro Phe Tyr Pro Asp Arg Met
Ala Gly Arg Ile 355 360 365Leu Gly Met Gly Asp Val Leu Ser Phe Val
Glu Lys Ala Gln Glu Val 370 375 380Met Lys Gln Glu Asp Ala Glu Asp
Leu Gln Lys Lys Ile Met Ser Ala385 390 395 400Lys Phe Asp Phe Asn
Asp Phe Leu Lys Gln Thr Arg Ala Val Ala Gln 405 410 415Met Gly Thr
Met Ser Arg Val Leu Gly Met Ile Pro Gly Met Gly Lys 420 425 430Val
Thr Pro Ala Gln Ile Arg Glu Ala Glu Lys Ser Leu Ile Ile Met 435 440
445Glu Ser Met Ile Glu Val Met Thr Pro Glu Glu Lys Glu Lys Pro Glu
450 455 460Leu Leu Ala Glu Ser Pro Ser Arg Arg Lys Arg Ile Ala Gln
Glu Ser465 470 475 480Gly Lys Thr Glu Gln Gln Val Ser Gln Leu Val
Ala Gln Leu Phe Gln 485 490 495Met Arg Val Arg Met Lys Asn Leu Met
Gly Val Met Gln Gly Gly Ser 500 505 510Ile Pro Ala Leu Ser Asn Leu
Glu Glu Ala Leu Lys Thr Glu Gln Lys 515 520 525Ala Pro Pro Gly Thr
Ala Arg Arg Lys Arg Arg Ser Glu Ser Arg Lys 530 535 540Gln Phe Ala
Asp Ser Gly Ser Thr Arg Pro Gly Pro Arg Gly Phe Gly545 550 555
560Ala Lys Asn211605DNANicotiana tabacum 21atgtccgcca ttgctacttc
tgctgctctc tcttttcctt tctctttctg ccgttctacc 60aagacttttg ctacaagaaa
gtgtttcaaa gggggatttg gagtgtttgc agtgtatgag 120gaggcaggtg
agttaacaaa caagaaaagc tcctggttga cactctttga tgtggaagat
180ccaaggtcaa aatttcctca gtctaaaggc aagttcctgg atgcaaatca
agctttagaa 240gttgctagat ttgatataca atattgtgat tggcgagctc
ggcaagatgt attaaccata 300atgctcctgc acgaaaaggt tgtggaagta
ttgaatcctc tggcacgtga gtacaaatct 360attggaacca tgaagaagga
actagcagag ttacaaggag cactttctca ggctcataaa 420gaggtacata
tatctgaggt gcgggtttct gctgctttag ataagctagc tcacatggaa
480gcattggtta atgataggct gcttccggag aggagtgcag aagaatcaga
ttgcccgtct 540tcctccaccg gtacgtctac agtatctaga gatactgtta
aaggcaagca gcctaggaga 600accctcaatg tgtcaggtcc ggtccaagat
tacagctctc atttgaagaa cttttggtat 660cctgtcgctt tttctgctga
tgttaaggaa gacacaatga caccaattga ttgctttgag 720gaaccatggg
tgatttttcg tgggaaagat ggaaaacctg gatgtgtccg gaacacatgt
780gcacatagag cctgccccct tcatttgggt tcagttaatg agggtcgcat
ccaatgtcct 840tatcatgggt gggaatattc aacagacgga aaatgtgaga
aaatgccatc aactaaattt 900ctgaatgtca agatcaaagc tctgccatgc
tttgagcaag agggaatgat atggatttgg 960cctggaaatg atcctcctgc
agctactctt ccttctttac tgccaccttc tggatttcaa 1020atccatgcag
agattgttat tgaacttcca gtggaacatg ggctactttt ggacaatctg
1080ttggatcttg cacatgctcc tttcacccat acgtctacat ttgctaaagg
atggactgtc 1140ccaagctttg taaaattttt gactcctgca tctggtcttc
aaggatattg ggatccctat 1200ccaatagata tggaatttcg accaccttgt
atagttctat caaccattgg aatctcaaag 1260ccaggcaagt tggaagggca
gagtaccaaa gagtgctcta cacacctaca ccaacttcat 1320gtatgtttac
ctgcatctaa acagaagaca aggttgttat ataggatgtc actggatttt
1380gctcccgtgc taaaacacat ccctttcatg caatacgtgt ggaggcattt
tgctgaacag 1440gttttaaacg aagacctacg gcttgtgatt ggtcagcaag
agcggatgct caatggtgct 1500aacatttgga acctgcctgt gtcatacgat
aagctaggag tgaggtatag gatatggaga 1560gatgctgtag agagtggagc
aaagcaattg ccattcagca aatga 160522534PRTNicotiana tabacum 22Met Ser
Ala Ile Ala Thr Ser Ala Ala Leu Ser Phe Pro Phe Ser Phe1 5 10 15Cys
Arg Ser Thr Lys Thr Phe Ala Thr Arg Lys Cys Phe Lys Gly Gly 20 25
30Phe Gly Val Phe Ala Val Tyr Glu Glu Ala Gly Glu Leu Thr Asn Lys
35 40 45Lys Ser Ser Trp Leu Thr Leu Phe Asp Val Glu Asp Pro Arg Ser
Lys 50 55 60Phe Pro Gln Ser Lys Gly Lys Phe Leu Asp Ala Asn Gln Ala
Leu Glu65 70 75 80Val Ala Arg Phe Asp Ile Gln Tyr Cys Asp Trp Arg
Ala Arg Gln Asp 85 90 95Val Leu Thr Ile Met Leu Leu His Glu Lys Val
Val Glu Val Leu Asn 100 105 110Pro Leu Ala Arg Glu Tyr Lys Ser Ile
Gly Thr Met Lys Lys Glu Leu 115 120 125Ala Glu Leu Gln Gly Ala Leu
Ser Gln Ala His Lys Glu Val His Ile 130 135 140Ser Glu Val Arg Val
Ser Ala Ala Leu Asp Lys Leu Ala His Met Glu145 150 155 160Ala Leu
Val Asn Asp Arg Leu Leu Pro Glu Arg Ser Ala Glu Glu Ser 165 170
175Asp Cys Pro Ser Ser Ser Thr Gly Thr Ser Thr Val Ser Arg Asp Thr
180 185 190Val Lys Gly Lys Gln Pro Arg Arg Thr Leu Asn Val Ser Gly
Pro Val 195 200 205Gln Asp Tyr Ser Ser His Leu Lys Asn Phe Trp Tyr
Pro Val Ala Phe 210 215 220Ser Ala Asp Val Lys Glu Asp Thr Met Thr
Pro Ile Asp Cys Phe Glu225 230 235 240Glu Pro Trp Val Ile Phe Arg
Gly Lys Asp Gly Lys Pro Gly Cys Val 245 250 255Arg Asn Thr Cys Ala
His Arg Ala Cys Pro Leu His Leu Gly Ser Val 260 265 270Asn Glu Gly
Arg Ile Gln Cys Pro Tyr His Gly Trp Glu Tyr Ser Thr 275 280 285Asp
Gly Lys Cys Glu Lys Met Pro Ser Thr Lys Phe Leu Asn Val Lys 290 295
300Ile Lys Ala Leu Pro Cys Phe Glu Gln Glu Gly Met Ile Trp Ile
Trp305 310 315 320Pro Gly Asn Asp Pro Pro Ala Ala Thr Leu Pro Ser
Leu Leu Pro Pro 325 330 335Ser Gly Phe Gln Ile His Ala Glu Ile Val
Ile Glu Leu Pro Val Glu 340 345 350His Gly Leu Leu Leu Asp Asn Leu
Leu Asp Leu Ala His Ala Pro Phe 355 360 365Thr His Thr Ser Thr Phe
Ala Lys Gly Trp Thr Val Pro Ser Phe Val 370 375 380Lys Phe Leu Thr
Pro Ala Ser Gly Leu Gln Gly Tyr Trp Asp Pro Tyr385 390 395 400Pro
Ile Asp Met Glu Phe Arg Pro Pro Cys Ile Val Leu Ser Thr Ile 405 410
415Gly Ile Ser Lys Pro Gly Lys Leu Glu Gly Gln Ser Thr Lys Glu Cys
420 425 430Ser Thr His Leu His Gln Leu His Val Cys Leu Pro Ala Ser
Lys Gln 435 440 445Lys Thr Arg Leu Leu Tyr Arg Met Ser Leu Asp Phe
Ala Pro Val Leu 450 455 460Lys His Ile Pro Phe Met Gln Tyr Val Trp
Arg His Phe Ala Glu Gln465 470 475 480Val Leu Asn Glu Asp Leu Arg
Leu Val Ile Gly Gln Gln Glu Arg Met 485 490 495Leu Asn Gly Ala Asn
Ile Trp Asn Leu Pro Val Ser Tyr Asp Lys Leu 500 505 510Gly Val Arg
Tyr Arg Ile Trp Arg Asp Ala Val Glu Ser Gly Ala Lys 515 520 525Gln
Leu Pro Phe Ser Lys 530231605DNANicotiana tabacum 23atgtccgcca
ttgctacttc tgctgctctc tcttttcctt tctctttttg ccgttctacc 60aagactttta
ctacaagaaa gtgtttcaaa gggggatttg gagtgtttgc agtgtatgag
120gaggcaggtg agttaacaaa caagaaaagc tcctggttga cactctttga
tgtggaagat 180ccaaggtcaa aatttcctca gtctaaaggc aagttcctgg
atgcaaatca agctttagaa 240gttgctagat ttgatatgca atattgtgat
tggcgagctc ggcaagacgt acttacaata 300atgctcctgc atgaaaaggt
tgtggaagta ttgaatcctc tagctcgtga atataaatct 360attggaacca
tgaagaagga actcgcggag ttacaagaag aactgtctcg ggctcacaaa
420gaggtacata tatctgaggt gcgggtttct gctgctttag ataagctagc
tcacatggaa 480gcattggtta atgataggct gcttccggag aggagtacag
aagaatcaga ttccccatct 540tcctccaccg gtacgtctac agtatctaga
gataatgcta aaagcaagca gcctaggaga 600accctcaatg tgtcaggtcc
cgtccaagat tacagctcct atttgaagaa cttttggtat 660cctgtggctt
tttctgctga tgttaaggaa gataccatga caccaattga ttgctttgag
720gaaccgtggg tgatttttcg tgggaaagat ggaaaacctg gatgtgtcca
gaacacatgt 780gcacatagag cttgccccct tcatttgggt tcagtgaatg
agggtcgcat acaatgtcct 840tatcacgggt gggaatattc aacagacgga
aaatgtgaga aaatgccatc aacaaaattt 900ctgaatgtca agatcaaagc
tctgccatgc tttgagcaag agggaatgat atggatttgg 960cctggaaacg
atcctcctgc agctactctt ccttctttgc taccaccttc tggatttcaa
1020atccatgcag agattgtcat ggaacttccg gtggaacatg ggctactttt
ggacaatctg 1080ttggatcttg cacatgctcc tttcactcat acgtctacat
ttgctaaagg atggactgtc 1140ccaagctttg taaaattttt gactcctgcg
tctggtctgc aaggatattg ggatccatat 1200ccaatagata tggaatttcg
accgccttgt atggttctgt caaccattgg aatctcaaag 1260ccgggcaaat
tggaagggca gagtatcaaa gagtgctcta cacaccttca ccaacttcat
1320gtatgtttac ctgcatctaa acagaagaca aggttgttat ataggatgtc
actggatttt 1380gctcctgttc taaaacacat ccctttcatg caatacgtgt
ggaggcattt tgctgaacag 1440gttttaaatg aagacctacg gcttgtgatt
ggtcagcaag aacggatgct caatggtgct 1500aacatttgga acctgcctgt
gtcatacgat aagctaggag tgaggtatag aatatggaga 1560gacgctgtag
agagtggagc aaagcagttg ccgttcagca aatga 160524534PRTNicotiana
tabacum 24Met Ser Ala Ile Ala Thr Ser Ala Ala Leu Ser Phe Pro Phe
Ser Phe1 5 10 15Cys Arg Ser Thr Lys Thr Phe Thr Thr Arg Lys Cys Phe
Lys Gly Gly 20 25 30Phe Gly Val Phe Ala Val Tyr Glu Glu Ala Gly Glu
Leu Thr Asn Lys 35 40 45Lys Ser Ser Trp Leu Thr Leu Phe Asp Val Glu
Asp Pro Arg Ser Lys 50 55 60Phe Pro Gln Ser Lys Gly Lys Phe Leu Asp
Ala Asn Gln Ala Leu Glu65 70 75 80Val Ala Arg Phe Asp Met Gln Tyr
Cys Asp Trp Arg Ala Arg Gln Asp 85 90 95Val Leu Thr Ile Met Leu Leu
His Glu Lys Val Val Glu Val Leu Asn 100 105 110Pro Leu Ala Arg Glu
Tyr Lys Ser Ile Gly Thr Met Lys Lys Glu Leu 115 120 125Ala Glu Leu
Gln Glu Glu Leu Ser Arg Ala His Lys Glu Val His Ile 130 135 140Ser
Glu Val Arg Val Ser Ala Ala Leu Asp Lys Leu Ala His Met Glu145 150
155 160Ala Leu Val Asn Asp Arg Leu Leu Pro Glu Arg Ser Thr Glu Glu
Ser 165 170 175Asp Ser Pro Ser Ser Ser Thr Gly Thr Ser Thr Val Ser
Arg Asp Asn 180 185 190Ala Lys Ser Lys Gln Pro Arg Arg Thr Leu Asn
Val Ser Gly Pro Val 195 200 205Gln Asp Tyr Ser Ser Tyr Leu Lys Asn
Phe Trp Tyr Pro Val Ala Phe 210 215 220Ser Ala Asp Val Lys Glu Asp
Thr Met Thr Pro Ile Asp Cys Phe Glu225 230 235 240Glu Pro Trp Val
Ile Phe Arg Gly Lys Asp Gly Lys Pro Gly Cys Val 245 250 255Gln Asn
Thr Cys Ala His Arg Ala Cys Pro Leu His Leu Gly Ser Val 260 265
270Asn Glu Gly Arg Ile Gln Cys Pro Tyr His Gly Trp Glu Tyr Ser Thr
275 280 285Asp Gly Lys Cys Glu Lys Met Pro Ser Thr Lys Phe Leu Asn
Val Lys 290 295 300Ile Lys Ala Leu Pro Cys Phe Glu Gln Glu Gly Met
Ile Trp Ile Trp305 310 315 320Pro Gly Asn Asp Pro Pro Ala Ala Thr
Leu Pro Ser Leu Leu Pro Pro 325 330 335Ser Gly Phe Gln Ile His Ala
Glu Ile Val Met Glu Leu Pro Val Glu 340 345 350His Gly Leu Leu Leu
Asp Asn Leu Leu Asp Leu Ala His Ala Pro Phe 355 360 365Thr His Thr
Ser Thr Phe Ala Lys Gly Trp Thr Val Pro Ser Phe Val 370 375 380Lys
Phe Leu Thr Pro Ala Ser Gly Leu Gln Gly Tyr Trp Asp Pro Tyr385 390
395 400Pro Ile Asp Met Glu Phe Arg Pro Pro Cys Met Val Leu Ser Thr
Ile 405 410 415Gly Ile Ser Lys Pro Gly Lys Leu Glu Gly Gln Ser Ile
Lys Glu Cys 420 425 430Ser Thr His Leu His Gln Leu His Val Cys Leu
Pro Ala Ser Lys Gln 435 440 445Lys Thr Arg Leu Leu Tyr Arg Met Ser
Leu Asp Phe Ala Pro Val Leu 450 455 460Lys His Ile Pro Phe Met Gln
Tyr Val Trp Arg His Phe Ala Glu Gln465 470 475 480Val Leu Asn Glu
Asp Leu Arg Leu Val Ile Gly Gln Gln Glu Arg Met 485 490 495Leu Asn
Gly Ala Asn Ile Trp Asn Leu Pro Val Ser Tyr Asp Lys Leu 500 505
510Gly Val Arg Tyr Arg Ile Trp Arg Asp Ala Val Glu Ser Gly Ala Lys
515 520 525Gln Leu Pro Phe Ser Lys 530251596DNANicotiana tabacum
25atgacagcca ttactactgc tctttctttt cctttctctt tgtgccgctc tactaagtct
60tatactagaa agtatgtcaa agggagcttt ggagtgtttg cagtatatgg ggaggagggt
120gggatgccag ataagaaaag ttcctggttg acactcttta atgtggaaga
tccaaggtct 180aaagttccac aaattaaagg caaattcttg gatgcaaatc
aagctttgga agttgctaga 240tatgatctac aatactgtga ttggcgagct
cggcaagatg tacttacaat catgctgctg 300catgaaaagg ttgtggaagt
gttgaaccct ctagcacgtg aatacaaatc tattggaacc 360atgaaaaagg
aacttgcaga gttgcaagga gagctttctc aggcccacaa ccaggtacat
420atatctgagg cccgggtttc tgctgctttg gataagctag cttacatgga
agagttggtt 480aatgataggc ttctgcaaga gagaagcacg gcagaatcag
attgctcgtc ctcctctgcc 540agtacgtcaa cagcattatt ggatactgtt
aaaagcaagc aaccccgaag aaccctgagt 600gtgtcaggtc ctgtccaaga
ttacagttcc cgtttgaaga acttttggta ccctgttgct 660ttctccgcag
atcttaagga tgacaccatg ttaccgattg attgctttga gcaaccatgg
720gtgatctttc gcgggaatga tggaaaacct ggatgtgtac agaatacgtg
tgcacataga 780gcctgccccc ttgatcttgg ctcagtgaaa gagggacgca
ttcagtgccc ttatcacgga 840tgggaatact caactgatgg gaagtgtgag
aaaatgccat caacacgatt actgaatgta 900aagatcaaag cactgccctg
ctttgagcaa gagggaatga tatggatttg gccaggaaat 960gatccccctg
cagctaccct tccttcttta ctaccgcctt ctggatttca aatccatgcg
1020gagatagtca tggaacttcc agtggaacat gggctattat tagacaattt
attggatctt 1080gcacatgctc ctttcaccca tacatcaacc tttgctaaag
gatggagtgt cccaagattg 1140gtgaagtttt tgactcctgc ttctggtctg
caaggatatt gggatcctta tccaatagat 1200atggaattta gaccaccttg
tatggtttta tcaaccattg gaatctcaaa gccaggcaaa 1260ttggaggggc
agagtaccaa gcagtgttgt acacaccttc atcaacttca tgtttgctta
1320cctgcatcac gacacaagac acggttatta tataggatgt cactggattt
tgctcccctg 1380ctgaaacaca tccctttcat gcaatatgtt tggagacatt
ttgccgaaca ggttttaaat 1440gaagacctac ggcttgtgtt gggccagcag
gatcgcatgc tcaatggcgc caatatttgg 1500aacttgccag tgtcttacga
taagctaggt gtgaggtata gaatatggag agatgctgta 1560gatagtggag
caaagcatct accattcagc aaataa 159626531PRTNicotiana tabacum 26Met
Thr Ala Ile Thr Thr Ala Leu Ser Phe Pro Phe Ser Leu Cys Arg1 5 10
15Ser Thr Lys Ser Tyr Thr Arg Lys Tyr Val Lys Gly Ser Phe Gly Val
20 25 30Phe Ala Val Tyr Gly Glu Glu Gly Gly Met Pro Asp Lys Lys Ser
Ser 35 40 45Trp Leu Thr Leu Phe Asn Val Glu Asp Pro Arg Ser Lys Val
Pro Gln 50 55 60Ile Lys Gly Lys Phe Leu Asp Ala Asn Gln Ala Leu Glu
Val Ala Arg65 70 75 80Tyr Asp Leu Gln Tyr Cys Asp Trp Arg Ala Arg
Gln Asp Val Leu Thr 85 90 95Ile Met Leu Leu His Glu Lys Val Val Glu
Val Leu Asn Pro Leu Ala 100 105 110Arg Glu Tyr Lys Ser Ile Gly Thr
Met Lys Lys Glu Leu Ala Glu Leu 115 120 125Gln Gly Glu Leu Ser Gln
Ala His Asn Gln Val His Ile Ser Glu Ala 130 135 140Arg Val Ser Ala
Ala Leu Asp Lys Leu Ala Tyr Met Glu Glu Leu Val145 150 155 160Asn
Asp Arg Leu Leu Gln Glu Arg Ser Thr Ala Glu Ser Asp Cys Ser 165 170
175Ser Ser Ser Ala Ser Thr Ser Thr Ala Leu Leu Asp Thr Val Lys Ser
180 185 190Lys Gln Pro Arg Arg Thr Leu Ser Val Ser Gly Pro Val Gln
Asp Tyr 195 200 205Ser Ser Arg Leu Lys Asn Phe Trp Tyr Pro Val Ala
Phe Ser Ala Asp 210 215 220Leu Lys Asp Asp Thr Met Leu Pro Ile Asp
Cys Phe Glu Gln Pro Trp225 230 235 240Val Ile Phe Arg Gly Asn Asp
Gly Lys Pro Gly Cys Val Gln Asn Thr 245 250 255Cys Ala His Arg Ala
Cys Pro Leu Asp Leu Gly Ser Val Lys Glu Gly 260 265 270Arg Ile Gln
Cys Pro Tyr His Gly Trp Glu Tyr Ser Thr Asp Gly Lys 275 280 285Cys
Glu Lys Met Pro Ser Thr Arg Leu Leu Asn Val Lys Ile Lys Ala 290 295
300Leu Pro Cys Phe Glu Gln Glu Gly Met Ile Trp Ile Trp Pro Gly
Asn305 310 315 320Asp Pro Pro Ala Ala Thr Leu Pro Ser Leu Leu Pro
Pro Ser Gly Phe 325 330 335Gln Ile His Ala Glu Ile Val Met Glu Leu
Pro Val Glu His Gly Leu 340 345 350Leu Leu Asp Asn Leu Leu Asp Leu
Ala His Ala Pro Phe Thr His Thr 355 360 365Ser Thr Phe Ala Lys Gly
Trp Ser Val Pro Arg Leu Val Lys Phe Leu 370 375 380Thr Pro Ala Ser
Gly Leu Gln Gly Tyr Trp Asp Pro Tyr Pro Ile Asp385 390 395 400Met
Glu Phe Arg Pro Pro Cys Met Val Leu Ser Thr Ile Gly Ile Ser 405 410
415Lys Pro Gly Lys Leu Glu Gly Gln Ser Thr Lys Gln Cys Cys Thr His
420 425 430Leu His Gln Leu His Val Cys Leu Pro Ala Ser Arg His Lys
Thr Arg 435 440 445Leu Leu Tyr Arg Met Ser Leu Asp Phe Ala Pro Leu
Leu Lys His Ile 450 455 460Pro Phe Met Gln Tyr Val Trp Arg His Phe
Ala Glu Gln Val Leu Asn465 470 475 480Glu Asp Leu Arg Leu Val Leu
Gly Gln Gln Asp Arg Met Leu Asn Gly 485 490 495Ala Asn Ile Trp Asn
Leu Pro Val Ser Tyr Asp Lys Leu Gly Val Arg 500 505 510Tyr Arg Ile
Trp Arg Asp Ala Val Asp Ser Gly Ala Lys His Leu Pro 515 520 525Phe
Ser Lys 53027723DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 27ggatccatgg cttctctatt
atcttctcgt ctcccacgtc atctttcctc taataaaccg 60gtactcccac catcaagctc
cggttcaaat ctccttcaca acttcacata taaaacccgg 120ttcgatcaat
cccggttcaa atgctcagct ggtggaacgg ggttcttcac gaagttgggt
180cgtttgctga aagagaaagc aaagagcgac gtggagaaac tgttctcagg
attctcaaaa 240actcgagaca atttagcagt tatagatgaa ctcctccttt
actggtaata agatcttcaa 300cacctacacc atttttttaa tcactactac
ccattgcatt gaacaaactt ccaagttctt 360cttagcttca gattaagaaa
gtaccctttc ttggctttgt tgatgtggta ccattgtcca 420ttgtcttgtg
tgtttccacc agtaaaggag gagttcatct ataactgcta aattgtctcg
480agtttttgag aatcctgaga acagtttctc cacgtcgctc tttgctttct
ctttcagcaa 540acgacccaac ttcgtgaaga accccgttcc accagctgag
catttgaacc gggattgatc 600gaaccgggtt ttatatgtga agttgtgaag
gagatttgaa ccggagcttg atggtgggag 660taccggttta ttagaggaaa
gatgacgtgg gagacgagaa gataatagag aagccattct 720aga
72328765DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 28ggatccggga aaggaatgga gtacttgata
gagtggaaag acgaacatgc cccaacgtgg 60gtcccctctg attacattgc taaagatgtt
gtggccgagt acgaaactcc ttggtggaat 120gcggctaaaa aggccgacga
atccgctctt agggaactcc tagaaactga cgacgacaga 180gatgtggacg
cagtagatga ggatggacgt acggctttgc tctttgtctc gggtctgggg
240tccgagccgt gtgtcaagct gctagctgaa gccggcgctg acgtggacta
tcgcgatagg 300aatggctaat aagatcttca acacctacac cattttttta
atcactacta cccattgcat 360tgaacaaact tccaagttct tcttagcttc
agattaagaa agtacccttt cttggctttg 420ttgatgtggt accattgtcc
attgtcttgt gtgtttccag ccattcctat cgcgatagtc 480cacgtcagcg
ccggcttcag ctagcagctt gacacacggc tcggacccca gacccgagac
540aaagagcaaa gccgtacgtc catcctcatc tactgcgtcc acatctctgt
cgtcgtcagt 600ttctaggagt tccctaagag cggattcgtc ggccttttta
gccgcattcc accaaggagt 660ttcgtactcg gccacaacat ctttagcaat
gtaatcagag gggacccacg ttggggcatg 720ttcgtctttc cactctatca
agtactccat tcctttccct ctaga 76529747DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
29ggatccatgg aagccactgc ttctttctcc tcaactatgt cttcccacca tttctttcca
60ctttccaaag ccaccctctc aacttctaaa cttccatttt ctgggactgg ttcaactcat
120tctctttcat tttcttcaag aaactcattc actagggata catggagggt
gatcaattca 180aggaatgtgg ttatttcaag aagagaaatg cgtggagtta
ttagagctga gatgtttgga 240cagctcacta gtggacttga atcagcttgg
aataagctca aaggagaaga ggttttgtaa 300taagatcttc aacacctaca
ccattttttt aatcactact acccattgca ttgaacaaac 360ttccaagttc
ttcttagctt cagattaaga aagtaccctt tcttggcttt gttgatgtgg
420taccattgtc cattgtcttg tgtgtttcca caaaacctct tctcctttga
gcttattcca 480agctgattca agtccactag tgagctgtcc aaacatctca
gctctaataa ctccacgcat 540ttctcttctt gaaataacca cattccttga
attgatcacc ctccatgtat ccctagtgaa 600tgagtttctt gaagaaaatg
aaagagaatg agttgaacca gtcccagaaa atggaagttt 660agaagttgag
agggtggctt tggaaagtgg aaagaaatgg tgggaagaca tagttgagga
720gaaagaagca gtggcttcca ttctaga 74730987DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
30ggatccgagg caggtgagtt aacaaacaag aaaagctcct ggttgacact ctttgatgtg
60gaagatccaa ggtcaaaatt tcctcagtct aaaggcaagt tcctggatgc aaatcaagct
120ttagaagttg ctagatttga tatacaatat tgtgattggc gagctcggca
agatgtatta 180accataatgc tcctgcacga aaaggttgtg gaagtattga
atcctctggc acgtgagtac 240aaatctattg gaaccatgaa aaaggaactt
gcagagttgc aaggagagct ttctcaggcc 300cacaaccagg tacatatatc
tgaggcccgg gtttctgctg ctttggataa gctagcttac 360atggaagagt
tggttaatga taggcttctg caagagagaa gcacggcaga atcagattaa
420taagatcttc aacacctaca ccattttttt aatcactact acccattgca
ttgaacaaac 480ttccaagttc ttcttagctt cagattaaga aagtaccctt
tcttggcttt gttgatgtgg 540taccattgtc cattgtcttg tgtgtttcca
atctgattct gccgtgcttc tctcttgcag 600aagcctatca ttaaccaact
cttccatgta agctagctta tccaaagcag cagaaacccg 660ggcctcagat
atatgtacct ggttgtgggc ctgagaaagc tctccttgca actctgcaag
720ttcctttttc atggttccaa tagatttgta ctcacgtgcc agaggattca
atacttccac 780aaccttttcg tgcaggagca ttatggttaa tacatcttgc
cgagctcgcc aatcacaata 840ttgtatatca aatctagcaa cttctaaagc
ttgatttgca tccaggaact tgcctttaga 900ctgaggaaat tttgaccttg
gatcttccac atcaaagagt gtcaaccagg agcttttctt 960gtttgttaac
tcacctgcct ctctaga 987313155DNAArtificial SequenceDescription of
Artificial Sequence Synthetic
polynucleotideMISC_FEATURE(1)..(480)pCSVMV
PromoterMISC_FEATURE(523)..(551)Cloning
regionMISC_FEATURE(601)..(840)NOS
terminatorMISC_FEATURE(841)..(1980)ACTII
PromoterMISC_FEATURE(2101)..(2820)NPT II Kan
ResistanceMISC_FEATURE(2941)..(3120)NOS Terminator 31aagcttccag
aaggtaatta tccaagatgt agcatcaaga atccaatgtt tacgggaaaa 60actatggaag
tattatgtga gctcagcaag aagcagatca atatgcggca catatgcaac
120ctatgttcaa aaatgaagaa tgtacagata caagatccta tactgccaga
atacgaagaa 180gaatacgtag aaattgaaaa agaagaacca ggcgaagaaa
agaatcttga agacgtaagc 240actgacgaca acaatgaaaa gaagaagata
aggtcggtga ttgtgaaaga gacatagagg 300acacatgtaa ggtggaaaat
gtaagggcgg aaagtaacct tatcacaaag gaatcttatc 360ccccactact
tatcctttta tatttttccg tgtcattttt gcccttgagt tttcctatat
420aaggaaccaa gttcggcatt tgtgaaaaca agaaaaaatt tggtgtaagc
tattttcttt 480gaagtactga ggatacaact tcagagaaat ttgtaagttt
gtggatcctg caggctagcg 540tgcactctag actcgacgaa ctgacgagct
cgaatttccc cgatcgttca aacatttggc 600aataaagttt cttaagattg
aatcctgttg ccggtcttgc gatgattatc atataatttc 660tgttgaatta
cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat
720gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa
aacaaaatat 780agcgcgcaaa ctatgataaa ttatcgcgcg cggtgtcatc
tatgttacta gatcgggaat 840tcctcgagca actattttta tgtatgcaag
agtcagcata tgtataattg attcagaatc 900gttttgacga gttcggatgt
agtagtagcc attatttaat gtacatacta atcgtgaata 960gtgaatatga
tgaaacattg tatcttattg tataaatatc cataaacaca tcatgaaaga
1020cactttcttt cacggtctga attaattatg atacaattct aatagaaaac
gaattaaatt 1080acgttgaatt gtatgaaatc taattgaaca agccaaccac
gacgacgact aacgttgcct 1140ggattgactc ggtttaagtt aaccactaaa
aaaacggagc tgtcatgtaa cacgcggatc 1200gagcaggtca cagtcatgaa
gccatcaaag caaaagaact aatccaaggg ctgagatgat 1260taattagttt
aaaaattagt taacacgagg gaaaaggctg tctgacagcc aggtcacgtt
1320atctttacct gtggtcgaaa tgattcgtgt ctgtcgattt taattatttt
tttgaaaggc 1380cgaaaataaa gttgtaagag ataaacccgc ctatataaat
tcatatattt tcctctccgc 1440tttgaattgt ctcgttgtcc tcctcacttt
catcagccgt tttgaatctc cggcgacttg 1500acagagaaga acaaggaaga
agactaagag agaaagtaag agataatcca ggagattcat 1560tctccgtttt
gaatcttcct caatctcatc ttcttccgct ctttctttcc aaggtaatag
1620gaactttctg gatctacttt atttgctgga tctcgatctt gttttctcaa
tttccttgag 1680atctggaatt cgtttaattt ggatctgtga acctccacta
aatcttttgg ttttactaga 1740atcgatctaa gttgaccgat cagttagctc
gattatagct accagaattt ggcttgacct 1800tgatggagag atccatgttc
atgttacctg ggaaatgatt tgtatatgtg aattgaaatc 1860tgaactgttg
aagttagatt gaatctgaac actgtcaatg ttagattgaa tctgaacact
1920gtttaaggtt agatgaagtt tgtgtataga ttcttcgaaa ctttaggatt
tgtagtgtcg 1980tacgttgaac agaaagctat ttctgattca atcagggttt
atttgactgt attgaactct 2040ttttgtgtgt ttgcagctca taaaaggtac
caaacaatga ttgaacaaga tggattgcac 2100gcaggttctc cggccgcttg
ggtggagagg ctattcggct atgactgggc acaacagaca 2160atcggctgct
ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
2220gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc
gcggctatcg 2280tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
acgttgtcac tgaagcggga 2340agggactggc tgctattggg cgaagtgccg
gggcaggatc tcctgtcatc tcaccttgct 2400cctgccgaga aagtatccat
catggctgat gcaatgcggc ggctgcatac gcttgatccg 2460gctacctgcc
cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
2520gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
cgcgccagcc 2580gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
aggatctcgt cgtgacccat 2640ggcgatgcct gcttgccgaa tatcatggtg
gaaaatggcc gcttttctgg attcatcgac 2700tgtggccggc tgggtgtggc
ggaccgctat caggacatag cgttggctac ccgtgatatt 2760gctgaagagc
ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
2820cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttttg
agcgggactc 2880tggcgatcgc cccgatcgtt caaacatttg gcaataaagt
ttcttaagat tgaatcctgt 2940tgccggtctt gcgatgatta tcatataatt
tctgttgaat tacgttaagc atgtaataat 3000taacatgtaa tgcatgacgt
tatttatgag atgggttttt atgattagag tcccgcaatt 3060atacatttaa
tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg
3120cgcggtgtca tctatgttac tagatcggga ctagt 31553220PRTNicotiana
tabacum 32Asn Gly Gly Gly Lys Thr Thr Ser Leu Gly Lys Leu Ala Asn
Arg Leu1 5 10 15Lys Lys Glu Gly 203323PRTNicotiana tabacum 33Arg
Gly Gly Cys Val Val Ser Val Val Asp Glu Leu Gly Ile Pro Val1 5 10
15Lys Phe Val Gly Val Gly Glu 203420PRTNicotiana tabacum 34Lys Arg
Gly Lys Gly Glu Asn Val Glu Tyr Leu Val Lys Trp Lys Asp1 5 10 15Gly
Glu Asp Asn 203522PRTNicotiana tabacum 35Arg Thr Ala Leu Leu Phe
Val Ser Gly Leu Gly Ser Glu Pro Cys Val1 5 10 15Lys Leu Leu Ala Glu
Ala 203620PRTNicotiana tabacum 36Arg Arg Ser Glu Ser Arg Lys Gln
Phe Ala Asp Ser Gly Ser Thr Arg1 5 10 15Pro Gly Pro Arg
203718PRTNicotiana tabacum 37Leu Lys Glu Val Lys Arg Val Leu Asn
Pro Thr Glu Val Leu Leu Val1 5 10 15Val Asp3827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38atggcttctc tattatcttc tcgtctc 273925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39gttcaaatgc tcagctggtg gaacg 254025DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
40cgtcaattcc tctctctccc gcctc 254125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
41cttcagaacc agcagcaaca agcag 254226DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
42atggaagcca ctgcttcttt ctcctc 264328DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
43ctcattcact agggatacat ggagggtg 28446PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
44His His His His His His1 545285DNAArtificial SequenceDescription
of Artificial Sequence Synthetic polynucleotide 45ggatccatgg
cttctctatt atcttctcgt ctcccacgtc atctttcctc taataaaccg 60gtactcccac
catcaagctc cggttcaaat ctccttcaca acttcacata taaaacccgg
120ttcgatcaat cccggttcaa atgctcagct ggtggaacgg ggttcttcac
gaagttgggt 180cgtttgctga aagagaaagc aaagagcgac gtggagaaac
tgttctcagg attctcaaaa 240actcgagaca atttagcagt tatagatgaa
ctcctccttt actgg 28546153DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 46taataagatc
ttcaacacct acaccatttt tttaatcact actacccatt gcattgaaca 60aacttccaag
ttcttcttag cttcagatta agaaagtacc ctttcttggc tttgttgatg
120tggtaccatt gtccattgtc ttgtgtgttt cca 15347285DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
47ccagtaaagg aggagttcat ctataactgc taaattgtct cgagtttttg agaatcctga
60gaacagtttc tccacgtcgc tctttgcttt ctctttcagc aaacgaccca acttcgtgaa
120gaaccccgtt ccaccagctg agcatttgaa ccgggattga tcgaaccggg
ttttatatgt 180gaagttgtga aggagatttg aaccggagct tgatggtggg
agtaccggtt tattagagga 240aagatgacgt gggagacgag aagataatag
agaagccatt ctaga 28548306DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 48ggatccggga
aaggaatgga gtacttgata gagtggaaag acgaacatgc cccaacgtgg 60gtcccctctg
attacattgc taaagatgtt gtggccgagt acgaaactcc ttggtggaat
120gcggctaaaa aggccgacga atccgctctt agggaactcc tagaaactga
cgacgacaga 180gatgtggacg cagtagatga ggatggacgt acggctttgc
tctttgtctc gggtctgggg 240tccgagccgt gtgtcaagct gctagctgaa
gccggcgctg acgtggacta tcgcgatagg 300aatggc
30649153DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
49taataagatc ttcaacacct acaccatttt tttaatcact actacccatt gcattgaaca
60aacttccaag ttcttcttag cttcagatta agaaagtacc ctttcttggc tttgttgatg
120tggtaccatt gtccattgtc ttgtgtgttt cca 15350306DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
50gccattccta tcgcgatagt ccacgtcagc gccggcttca gctagcagct tgacacacgg
60ctcggacccc agacccgaga caaagagcaa agccgtacgt ccatcctcat ctactgcgtc
120cacatctctg tcgtcgtcag tttctaggag ttccctaaga gcggattcgt
cggccttttt 180agccgcattc caccaaggag tttcgtactc ggccacaaca
tctttagcaa tgtaatcaga 240ggggacccac gttggggcat gttcgtcttt
ccactctatc aagtactcca ttcctttccc 300tctaga 30651297DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
51ggatccatgg aagccactgc ttctttctcc tcaactatgt cttcccacca tttctttcca
60ctttccaaag ccaccctctc aacttctaaa cttccatttt ctgggactgg ttcaactcat
120tctctttcat tttcttcaag aaactcattc actagggata catggagggt
gatcaattca 180aggaatgtgg ttatttcaag aagagaaatg cgtggagtta
ttagagctga gatgtttgga 240cagctcacta gtggacttga atcagcttgg
aataagctca aaggagaaga ggttttg 29752153DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
52taataagatc ttcaacacct acaccatttt tttaatcact actacccatt gcattgaaca
60aacttccaag ttcttcttag cttcagatta agaaagtacc ctttcttggc tttgttgatg
120tggtaccatt gtccattgtc ttgtgtgttt cca 15353297DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
53caaaacctct tctcctttga gcttattcca agctgattca agtccactag tgagctgtcc
60aaacatctca gctctaataa ctccacgcat ttctcttctt gaaataacca cattccttga
120attgatcacc ctccatgtat ccctagtgaa tgagtttctt gaagaaaatg
aaagagaatg 180agttgaacca gtcccagaaa atggaagttt agaagttgag
agggtggctt tggaaagtgg 240aaagaaatgg tgggaagaca tagttgagga
gaaagaagca gtggcttcca ttctaga 29754417DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
54ggatccgagg caggtgagtt aacaaacaag aaaagctcct ggttgacact ctttgatgtg
60gaagatccaa ggtcaaaatt tcctcagtct aaaggcaagt tcctggatgc aaatcaagct
120ttagaagttg ctagatttga tatacaatat tgtgattggc gagctcggca
agatgtatta 180accataatgc tcctgcacga aaaggttgtg gaagtattga
atcctctggc acgtgagtac 240aaatctattg gaaccatgaa aaaggaactt
gcagagttgc aaggagagct ttctcaggcc 300cacaaccagg tacatatatc
tgaggcccgg gtttctgctg ctttggataa gctagcttac 360atggaagagt
tggttaatga taggcttctg caagagagaa gcacggcaga atcagat
41755153DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 55taataagatc ttcaacacct acaccatttt
tttaatcact actacccatt gcattgaaca 60aacttccaag ttcttcttag cttcagatta
agaaagtacc ctttcttggc tttgttgatg 120tggtaccatt gtccattgtc
ttgtgtgttt cca 15356417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 56atctgattct
gccgtgcttc tctcttgcag aagcctatca ttaaccaact cttccatgta 60agctagctta
tccaaagcag cagaaacccg ggcctcagat atatgtacct ggttgtgggc
120ctgagaaagc tctccttgca actctgcaag ttcctttttc atggttccaa
tagatttgta 180ctcacgtgcc agaggattca atacttccac aaccttttcg
tgcaggagca ttatggttaa 240tacatcttgc cgagctcgcc aatcacaata
ttgtatatca aatctagcaa cttctaaagc 300ttgatttgca tccaggaact
tgcctttaga ctgaggaaat tttgaccttg gatcttccac 360atcaaagagt
gtcaaccagg agcttttctt gtttgttaac tcacctgcct ctctaga 417
* * * * *